The androgen receptor (AR) is a sequence-specific DNA-binding protein that plays a key role in prostate cancer cellular proliferation by dihydrotestosterone and the induction of secondary sexual characteristics.In this study we demonstrate that the AR can be modified by acetylation in vitro and in vivo. p300 and p300/ cAMP-response element-binding protein acetylated the AR at a highly conserved lysine-rich motif carboxylterminal to the zinc finger DNA-binding domain.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ER␣) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ER␣-regulated gene expression involves interactions with cointegrators (e.g. p300/ CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ER␣ is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ER␣ at lysine residues within the ER␣ hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ER␣ hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ER␣ acetylation normally suppresses ligand sensitivity. These ER␣ lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ER␣ acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

Anti-dsDNA Abs are characteristic of lupus and can be found deposited in the kidneys of lupus mice. Previously, we have shown that pathogenic anti-dsDNA Abs as well as Ig eluted from the kidneys of nephritic lupus mice cross-react with α-actinin. Moreover, cross-reactivity with α-actinin characterizes nephritogenic anti-dsDNA Abs in humans with lupus as well. To determine whether Abs generated against α-actinin in vivo cross-react with nuclear Ags, we s.c. immunized 10-wk-old female BALB/c mice (and several other nonautoimmune mice strains) with α-actinin in adjuvant. Immunized but not control mice displayed high titers of anti-nuclear Abs and IgG anti-chromatin autoantibodies, hypergammaglobulinemia, renal Ig deposition, and proteinuria. The specificity of the anti-chromatin response was determined by Western blotting of purified chromatin with serum from α-actinin immunized mice. By proteomic analysis, a 25-kDa doublet band was conclusively identified as high mobility group box (HMGB) proteins 1 and 3, and a 70-kDa band was identified as heat shock protein 70 (hsp70), both of which are known antigenic targets in murine lupus. Binding to purified HMGB1 and hsp70 by immunized mice sera was confirmed by ELISA and Western blot. Immunized mice sera binding to both 25- and 70-kDa bands were significantly inhibited by α-actinin and chromatin. Importantly, a panel of nephritogenic mAbs had significantly higher affinity for α-actinin, chromatin, HMGB, and hsp70 as compared with nonpathogenic Abs, suggesting a common motif in these Ags that is targeted by pathogenic autoantibodies.

Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx. 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties [Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346]. In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability. Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing. Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues. The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme. Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit. Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information. The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da. Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme [Huang and Colman (1990) Biochemistry 29, 8266-8273] suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme. An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated. One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated. Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known [Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316] shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function.

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