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- Gaullier, Guillaume, et al.
(författare)
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A higher-order entity formed by the flexible assembly of RAP1 with TRF2.
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:4, s. 1962-76
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Tidskriftsartikel (refereegranskat)abstract
- Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.
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| 4. |
- Kozlenkov, Alexey, et al.
(författare)
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Residues determining the specific binding of uncompetitive inhibitors to mammalian alkaline phosphatases
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Recent data have suggested that the activity of human tissue-nonspecific alkaline phosphatase (TNAP) could be targeted therapeutically to ameliorate soft-tissue ossification abnormalities resulting from insufficient production of inorganic pyrophosphate (1). Thus, understanding the mechanism of action and precise binding site for inhibitors of TNAP function is paramount. We compared the modeled three-dimensional structure of TNAP with the 3D structure of human placental alkaline phosphatase (PLAP), and identified the residues that differ between these two isozymes within a 12 Å radius of the active site. We then used site-directed mutagenesis to substitute TNAP residues to their respective homologues in PLAP or in the related germ cell (GCAP) isozyme or to Ala. In addition, we mutagenized most of the corresponding residues in PLAP to their TNAP homologues, and studied the role of a conserved residue Y371 in TNAP using the A371 mutation. All mutants were characterized for their sensitivity towards the uncompetitive inhibitors Lhomoarginine (L-hArg), levamisole, theophylline and L-phenylalanine. We found that the identity of residue108 in TNAP largely determines the specificity of inhibition by LhArg. The conserved Tyr-371 is also necessary for binding of L-hArg. The selectivity of inhibition by L-Phe was determined by the identity of residues 108 and 109 in TNAP (or corresponding residues 107 and 108 in PLAP). In contrast, the binding of levamisole to TNAP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. Substitutions H434E (as in PLAP), H434Q (as in chicken TNAP), H434S (as in the intestinal isozyme), H434G (as in GCAP), or H434A, all lead to a significant decrease in inhibition. The reciprocal E429H mutation in PLAP improved the inhibition by levamisole by 10-fold. The main determinant of sensitivity to theophylline is His-434. The H434E substitution (as in PLAP) causes a 50-fold reduction in inhibition, making TNAP even less inhibited than wt PLAP. The reciprocal E429H mutation in PLAP improves the Ki for theophylline inhibition 30 times, close to the level of wt TNAP. Interestingly, theophylline inhibition in TNAP could be further improved by introducing an E108F mutation. Thus, we have clarified the location of the binding sites for all three TNAP inhibitors and we have also been able to exchange inhibitor specificities between TNAP and PLAP. These data will help us in drug design efforts aimed at discovering novel and better inhibitors of TNAP for clinical use.
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- Llinas, Paola, et al.
(författare)
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Structural studies of human alkaline phosphatase in complex with strontium : implication for its secondary effect in bones.
- 2006
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 15:7, s. 1691-700
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Tidskriftsartikel (refereegranskat)abstract
- Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.
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