The binding of nonspecific DNA to the C-terminal negative regulatory domain (CTD) of p53 modulates its activity. The CTD is a natively unfolded region, which is subject to acetylation and phosphorylation at several residues as part of control. To measure the effect of covalent modification on binding to DNA, we synthesized a series of fluorescein-labeled CTD peptides with single and multiple acetylations at lysine residues that we had identified by NMR as making contact with DNA, and developed an analytical ultracentrifugation method to study their binding to DNA. Binding depended on ionic strength, indicating an electrostatic contribution. Monoacetylation weakened DNA binding at physiological ionic strength 2- to 3-fold, diacetylations resulted in further 2- to 3-fold decrease in the affinity, and tri- and tetraacetylations rendered DNA binding undetectable. Phosphorylation at S392 did not affect DNA binding. NMR spectroscopy showed binding to DNA did not induce significant structure into CTD, apart possibly from local helix formation.

1. Protein synthesis has been investigated in different regions of the rat epididymis by measuring incorporation of [35S]methionine in tissue minces incubated in vitro followed by analysis of labelled proteins on polyacrylamide gels containing sodium dodecyl sulphate. Rates of synthesis were highest in the proximal cauda > distal cauda > initial segment > ductuli efferentes > corpus > distal caput > proximal caput. One protein (mol.wt. 23 000) characterized the initial segment, three proteins (mol.wts. 18 500, 19 000 and 32 000) the caput and one protein (mol.wt. 47 000) the cauda. 2. After castration, [35S]methionine incorporation in all regions of the epididymis was reduced to < 10% of that in normal animals but could be restored to control levels within 5 days by testosterone treatment. Other steroids (corticosterone, oestrogen or progesterone) were ineffective. 3. The synthesis of the 18 500, 19 000, and 32 000 mol.wt. proteins in the caput and the 47 000 mol.wt. protein in the cauda were preferentially regulated by androgens, whilst the synthesis of 23 000 and approx. 80 000 mol.wt. proteins in the initial segment was dependent upon factors present in testicular fluid. 4. The androgen-dependent and testicular fluid-dependent proteins were major components of epididymal secretion. Purification and characterization of the 18 500, 19 000, 23 000 and 32 000 mol.wt. proteins showed them to be acidic glycoproteins with a carbohydrate content of 7.6-13.2%. The 47 000 mol.wt. protein, on the other hand, is highly basic. 5. A possible role for these proteins in the acquisition of motility, fertilizing capacity and storage of spermatozoa in the epididymis is discussed.

Homologous recombination is repressed by the binding of p53 to Rad51. We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179 -190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site. The p53 site is quite promiscuous, since it also binds peptides derived from 53BP1, 53BP2, Hif-1␣, and BCL-X L in overlapping regions. Binding is mediated mainly by a strong, nonspecific, electrostatic component and is fine tuned by specific interactions. Competition of the different proteins with each other and with specific DNA for a single site in p53 could be a factor in regulation of its activity.

Glycoproteins on the plasma membrane of testicular and cauda epididymidal spermatozoa have been labeled with galactose oxidase/NaB [aH]4 and sodium metaperiodate/ NaB[3H]4, followed by analysis on SDS polyacrylamide gels. The major glycoprotein labeling on testicular spermatozoa has a molecular weight 110,000 whereas on cauda epididymidal spermatozoa >90% of the radio-label is incorporated into proteins of molecular weight 32,000. These 32,000-mol wt proteins are homologous with proteins of similar molecular weight purified from the epididymal secretion and which have been shown previously to be synthesized in the caput epididymidis under hormonal control. Immunofluorescence revealed that the 32,000-mol wt proteins are present on the flagellum of mature but not immature spermatozoa and that they have a patchy distribution suggesting that they are mobile within the plane of the membrane. The membrane-bound 32,000-mol wt proteins possess hydrophobic domains as revealed by charge-shift electrophoresis and they also label with a lipophilic photoaffinity probe suggesting that they are in contact with the lipid bilayer. The evidence indicates that there is a considerable reorganization of the molecular structure of the plasma membrane of spermatozoa during maturation in the epididymis and that some of the changes are brought about by a direct interaction with epididymal secretory proteins.The acquisition of motility and fertilizing capacity by mammalian spermatozoa as they pass through the epididymis is accompanied by a number of distinct, albeit in some cases subtle, changes in their morphology, composition, and metabohc activity (reviewed in references 2 and 42). That the epididymis plays a significant role in regulating sperm maturation is not in doubt, but relatively little is known about the processes mediating or controlling these changes, the sequence in which they take place, and their relative importance to the ultimate ability of the sperm to fertilize an egg. Recently, interest has focussed on maturation changes in the plasma membrane as the initial events during fertilization involve cellular recognition and membrane fusion. The concept that the plasma membrane of mature spermatozoa is a mosaic of heterogeneous protein and lipid domains has arisen from studies on the binding of lectins (31), distribution of antigens (1, 11,15,16,25,28,30,33), surface charge (21,37,50), and the arrangement ofintramembrane particles (12,29,32). However, it is not altogether clear from these studies whether this heterogeneity is already expressed on spermatozoa in the testis or if it arises by modification of the plasma membrane during maturation in the epididymis.As a preliminary step towards investigating the role of the epididymis in regulating sperm maturation we have examined the synthesis and secretion of proteins and glycoproteins in the rat epididymis (8,26). Several androgen-dependent proteins and one testicular fluid-dependent protein were identified and characterized from the luminal secretions. In this c...

No abstract