ABSTRACT
Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)(n) repeats, and though similar repeated sequences-(NTT)(n), (DTT)(n), (ETT)(n), (NSS)(n), (ESS)(n), (DAA)(n), (ASS)(n), and (NAA)(n)-also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)(n) peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.
Subject(s)
Dentin/drug effects , Extracellular Matrix Proteins/chemistry , Peptides/pharmacology , Phosphoproteins/chemistry , Sialoglycoproteins/chemistry , Tooth Calcification/drug effects , Tooth/drug effects , Amino Acid Sequence/physiology , Animals , Binding Sites/drug effects , Binding Sites/physiology , Cells, Cultured , Dental Caries/diagnosis , Dental Caries/metabolism , Dental Caries/physiopathology , Dentin/growth & development , Dentin/metabolism , Durapatite/chemistry , Durapatite/metabolism , Extracellular Matrix Proteins/metabolism , Mice , Peptides/metabolism , Peptides/therapeutic use , Phosphoproteins/metabolism , Phosphorylation , Protein Binding/drug effects , Protein Binding/physiology , Sialoglycoproteins/metabolism , Tooth/growth & development , Tooth/metabolism , Tooth Calcification/physiologyABSTRACT
The cAMP receptor protein (Crp) is a global transcriptional regulator that binds sequence-specific promoter elements when associated with cAMP. In the motile cyanobacterium Synechocystis sp. strain PCC 6803, intracellular cAMP increases when dark-adapted cells are illuminated. Previous work has established that Crp binds proposed Crp target sites upstream of slr1351 (murF), sll1874 (chlA(II)), sll1708 (narL), slr0442 and sll1268 in vitro, and that slr0442 is downregulated in a crp mutant during photoautotrophic growth. To identify additional Crp target genes in Synechocystis, 11 different Crp binding sites proposed during a previous computational survey were tested for in vitro sequence-specific binding and crp-dependent transcription. The results indicate that murF, chlA(II) and slr0442 can be added as 'target genes of Sycrp1' in Synechocystis. Promoter mapping of the targets revealed the same close association of RNA polymerase and Crp as that found in Escherichia coli class I and class II Crp-regulated promoters, thereby strongly suggesting similar mechanisms of transcriptional activation.
