An Atypical Form of αB-crystallin Is Present in High Concentration in Some Human Cataractous Lenses IDENTIFICATION AND CHARACTERIZATION OF ABERRANT N- AND C-TERMINAL PROCESSING

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, αB-crystallin. The concentration of total αB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as αBg. Mass spectrometric analyses of tryptic and Asp-N digests showed αBg is αB-crystallin minus the C-terminal lysine. αBg constituted 10–90% of the total αB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of αB-crystallin. Human αBg and αB-crystallin were cloned and expressed inEscherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human αBg was comparable to that of recombinant human αB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating αBg is not known, but a premature termination of the αB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the αB-crystallin gene from lenses containing αBg. The 16.4-kDa protein was an N-terminally truncated fragment of αBg. The high concentration of αB-crystallin in these cataracts is the first observation of this kind in human lenses

PhD

dissertationSubstantial biochemical and genetic evidence exists that implicates ribosomal RNA as a functional component of the translational apparatus. The nucleotide sequences in functionally important regions of ribosomal RNA have defined features of universal conservation, single-stranded ness [as defined by secondary structure], and surface proximity [as defined by tertiary or quaternary structure]. Specific nucleotides within these regions are posttranscriptionally modified, yet little in known about the chemical identity of these modifications, and almost nothing is known about their function. This paucity of information stems from the lack of practical methodologies required to elucidate chemical structure of posttranscriptionally modified nucleotides as a prerequisite to investigation of structure/function relationships. A method for the detection, chemical characterization, and sequence location of posttranscriptionally modified nucleotides in RNA is presented. The method is based on the ability to infer the base composition of an oligonucleotide, simple by accurate measurement of molecular mass by electrospray mass spectrometry. Posttranscriptional modifications are recognized from incremental increases in mass, In conjunction with combined liquid chromatography/thermospray mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level and assigned to sequence sites within oligonucleotides defined by selective ribonuclease cleavage. The method is demonstrated using Escherichia coli 5S rRNA, in which all oligonucleotides produced by ribonuclease T1 hydrolysis are identified solely on the basis of their molecular masses, and using Escherichia coli 16S rRNA, in which several posttranscriptionally modified oligonucleotides were characterized. The method is used to characterized, 5,6-dihydrouridine at position 2449 in the highly conserved central loop of domain V in Escherichia coli 23S ribosomal RNA, a region which has been strongly implicated in the peptidyl transferase activity

Regulation of α-Synuclein Expression by Poly (ADP Ribose) Polymerase-1 (PARP-1) Binding to the NACP-Rep1 Polymorphic Site Upstream of the SNCA Gene

Alleles at NACP-Rep1, the polymorphic microsatellite repeat located ∼10 kb upstream of the α-synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5–2-fold increase in α-synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1’s catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1’s catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression