Raymond W. Ruddon

Selective Cytotoxicity to Human Leukemic Myeloblasts Produced by Oligodeoxyribonucleotide Phosphorothioates Complementary to p53 Nucleotide Sequences

Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.

Cystine Knot Mutations Affect the Folding of the Glycoprotein Hormone α-Subunit DIFFERENTIAL SECRETION AND ASSEMBLY OF PARTIALLY FOLDED INTERMEDIATES

The common glycoprotein hormone α-subunit (GPH-α) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10–60, 28–82, and 32–84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-α folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin β-subunit (hCG-β). Mutation of non-cystine knot disulfide bond 7–31, bond 59–87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-β. From these studies we conclude that: (i) the cystine knot of GPH-α is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-α folding, its ability to be secreted, and its ability to heterodimerize with hCG-β.

Human chorionic gonadotropin-beta gene expression in first trimester placenta.

The hCGβ gene family contains six genes linked in tandem on chromosome 19 and labeled β genes 7, 8, 5, 1, 2, and 3. Previous studies on a small number of placentas have indicated that β gene 5 was the most highly expressed gene during the first trimester of pregnancy, followed by genes 3 and 8. β genes 7, 1, and 2 were expressed at very low levels. The purpose of this study was to determine 1) whether this pattern of expression was typical during normal pregnancy by sampling a large number of first trimester placentas, and 2) whether there was a correlation between gestational age and the pattern of hCGβ gene expression. Total RNA from 27 first trimester placentas varying in age from 6–16 weeks was reverse transcribed into complementary DNA. The complementary DNA was amplified by PCR, and the amount of DNA representative of each hCGβ gene was quantified by Genescan analysis. In 14 of the 27 placentas, hCGβ gene 5 accounted for 50% or more of the total β messenger RNA expressed.β gene 3 was expressed at le...

A linear 23-residue peptide reveals a propensity to form an unusual native-like conformation.

To gain insight into the earliest events of protein folding, a 23-residue peptide with a sequence corresponding to the 38-60 fragment of the beta-subunit of human chorionic gonadotropin (hCG beta) was studied by NMR. In aqueous solution the majority of the peptide residues adopted an extended polyproline II (PII) conformation similar to those in mature, fully folded hCG beta. The finding that the isolated protein fragment may acquire native-like structural motifs, even without alpha-helices or beta-structures, extends the possibility of using free peptides as model systems to better understand the protein folding mechanisms. It was shown that the PII-rich structural motif can be determined efficiently by NMR spectroscopy. The observation that in the absence of extensive medium- and long-range interactions the majority of amino acid residues may adopt the PII conformation suggests that the PII-rich structural motifs may play an important role in early events of protein folding.

Folding of the β-Subunit of hCG and Its Role in Assembly of the α-β Heterodimer

It has been known for some time that the assembly of the α- and β-subunits of human chorionic gonadotropin (hCG) within cells that produce it is not totally efficient. As a result, uncombined α- and β-subunits, as well as α-β heterodimer, are secreted (1–3). Efficiency of α-β assembly in vitro, using purified urinary forms of the subunits, has also been reported to be low, with a kd of 0.6 μM (4).