Showing papers by "Charles P. Ordahl published in 2004"

Regulation of the enzymatic catalysis of poly(ADP-ribose) polymerase by dsDNA, polyamines, Mg2+, Ca2+, histones H1 and H3, and ATP.

Abstract: The enzymatic mechanism of poly(ADP-ribose) polymerase (PARP-1) has been analyzed in two in vitro systems: (a) in solution and (b) when the acceptor histones were attached to a solid surface. In system (a), it was established that the coenzymatic function of dsDNAs was sequence-independent. However, it is apparent from the calculated specificity constants that the AT homopolymer is by far the most effective coenzyme and randomly damaged DNA is the poorest. Rates of auto(poly-ADP-ribosylation) with dsDNAs as coenzymes were nearly linear for 20 min, in contrast to rates with dcDNA, which showed product [(ADPR)n] inhibition. An allosteric activation of auto(poly-ADP-ribosylation) by physiologic cellular components, Mg2+, Ca2+, and polyamines, was demonstrated, with spermine as the most powerful activator. On a molar basis, histones H1 and H3 were the most effective PARP-1 activators, and their action was abolished by acetylation of lysine end groups. It was shown in system (b) that oligo(ADP-ribosyl) transf...

Clathrin Isoform CHC22, a Component of Neuromuscular and Myotendinous Junctions, Binds Sorting Nexin 5 and Has Increased Expression during Myogenesis and Muscle Regeneration

Abstract: The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

Analysis of nucleotide sequence-dependent DNA binding of poly(ADP-ribose) polymerase in a purified system

Abstract: The enzymatic transfer of ADP-ribose from NAD to histone H(1) [defined as trans(oligo-ADP-ribosylation)] or to PARP-1 [defined as auto(poly-ADP-ribosylation)] requires binding of coenzymic DNA. The preceding paper [Kun, E., et al. (2004) Biochemistry 43, 210-216] shows that oligonucleotides of dsDNA can serve as coenzymic DNA for PARP-1 trans- or auto-modification activity. Results of DNA-protein binding (EMSA) experiments reported here demonstrate that short DNA oligonucleotides containing the 5'-TGTTG-3' nucleotide sequence motif preferentially bind to cloned PARP-1 in vitro. The same nucleotide sequence motif is responsible for striated myocyte-selective transcription of a contractile protein gene encoding cardiac troponin T (cTnT). Results of experiments reported here demonstrate that mutation of this motif also abolishes the differentiation-dependent activation of the transfected cTnT promoter in myoblasts cultured in vitro, indicating that nucleotide sequence-dependent binding of PARP-1 to promoter DNA of the cTnT gene is also necessary for differentiation-dependent activation. Thus, PARP-1 has two types of dsDNA binding activity: (1) nucleotide sequence-dependent binding, analyzed here with EMSA experiments, and (2) coenzymic binding, measured catalytically, which does not depend on the nucleotide sequence of the dsDNA. We hypothesize that the well-known association of PARP-1 with chromatin can be attributed to its stable binding to chromosomal dsDNA, some portion of which is likely to be nucleotide sequence-dependent binding. According to this hypothesis, the distribution of this protein-modifying enzyme in chromatin may be targeted to specific genomic loci and vary according to cell type and developmental stage.

Precocious terminal differentiation of premigratory limb muscle precursor cells requires positive signalling

Abstract: The timing of myogenic differentiation of hypaxial muscle precursor cells in the somite lags behind that of epaxial precursors. Two hypotheses have been proposed to explain this delay. One attributes the delay to the presence of negative-acting signals from the lateral plate mesoderm adjacent to the hypaxial muscle precursor cells located in the ventrolateral lip of the somitic dermomyotome (Pourquie et al. [1995] Proc. Natl. Acad. Sci. USA 92:3219-3223). The second attributes the delay to an absence of positive-acting inductive signals, similar to those from the axial structures that induce epaxial myotome development (Pownall et al. [1996] Development 122:1475-1488). Because both studies relied principally upon changes in the expression pattern of mRNAs specific to early muscle precursor cell markers, we revisited these experiments using two methods to assess muscle terminal differentiation. First, injection of fluorescent dyes before surgery was used to determine whether ventrolateral lip cells transform from epithelial cells to elongated myocytes. Second, an antibody to a terminal differentiation marker and a new monoclonal antibody that recognises avian and mammalian Pax3 were used for immunohistochemistry to assess the transition from precursor cell to myocyte. The results support both hypotheses and show further that placing axial structures adjacent to the somite ventrolateral lip induces an axial pattern of myocyte terminal differentiation and elongation.