The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation

Knowledge about the sizes of peptides generated by proteasomes during protein degradation is essential to fully understand their degradative mechanisms and the subsequent steps in protein turnover and generation of major histocompatibility complex class I antigenic peptides. We demonstrate here that 26 S and activated 20 S proteasomes from rabbit muscle degrade denatured, nonubiquitinated proteins in a highly processive fashion but generate different patterns of peptides (despite their containing identical proteolytic sites). With both enzymes, products range in length from 3 to 22 residues, and their abundance decreases with increasing length according to a log-normal distribution. Less than 15% of the products are the length of class I presented peptides (8 or 9 residues), and two-thirds are too short to function in antigen presentation. Surprisingly, these mammalian proteasomes, which contain two "chymotryptic," two "tryptic," and two "post-acidic" active sites, generate peptides with a similar size distribution as do archaeal 20 S proteasomes, which have 14 identical sites. Furthermore, inactivation of the "tryptic" sites altered the peptides produced without significantly affecting their size distribution. Therefore, this distribution is not determined by the number, specificity, or arrangement of the active sites (as proposed by the "molecular ruler" model); instead, we propose that proteolysis continues until products are small enough to diffuse out of the proteasomes.     

The Glu318Gly mutation of the presenilin-1 gene does not necessarily cause Alzheimer's disease

In early-onset familial Alzheimer's disease (AD) pathogenic mutations have been found in the amyloid precursor protein (APP) gene and in the presenilin (PS)-1 and PS-2 genes. We screened for mutations in these genes in 20 patients with familial AD from the Finnish population. In addition, we sampled 41 sporadic AD patients and 59 controls to test for mutations identified in our familial AD cases. We detected an A-to-G transition in the PS-1 gene, resulting in a glutamic acid (Glu)-to-glycine (Gly) substitution at codon 318 in 2 familial and 2 sporadic AD patients. The Glu318Gly mutation has previously been reported to cause AD. We also found the Glu318Gly mutation in 4 healthy aged controls (range, 74-87 years). We thus conclude that the mutation is most likely a rare polymorphism not related to AD.     

Testosterone and testosterone precursors in the spermatic vein and in the testicular tissue of old men. Reduced oxygen supply may explain the relative increase of testicular progesterone and 17 alpha-hydroxyprogesterone content and production in old age

Testosterone and the testosterone precursors pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, androstenedione, androstenediol and dehydroepiandrosterone were measured in the spermatic vein plasma and in the testicular tissue of young and old men. Testosterone and its precursors decreased in the testicular tissue of old men. However, progesterone and 17 alpha-hydroxyprogesterone increased in relation to testosterone in the testicular tissue and in the spermatic vein of old men. It is assumed that these age-dependent changes are caused by an impaired oxygen supply of the ageing testes. This hypothesis is supported by the observation that the same changes in steroid pattern seen in old age can be observed under reduced oxygen supply in in vitro incubation experiments with testicular tissue.     

Hormone changes in relation to the time of fetal death after prostaglandin-induced abortion

The changes in unconjugated estradiol-17beta and estriol, progesterone and chorionic somatomammotropin (HCS) in peripheral plasma have been studied in 18 women at 30-minute intervals following intra-uterine prostaglandin E2 administration for therapeutic termination of second trimester pregnancy. The hormonal changes were related to the time of fetal death detected by the disappearance of fetal heart pulsations. Prostaglandin E2 was given by the intra-amniotic route with urea (5 patients) or with intravenous oxytocin (5 patients), or by the extra-amniotic route with intravenous oxytocin (8 patients). Fetal death occurred rapidly with intra-amniotic PGE2, but usually at a late stage with extra-amniotic PGE2. Three fetuses in the extra-amniotic group died at or just before abortion. A variety of fetal heart changes were noted and the time of fetal death did not appear to influence the time of abortion within each treatment subgroup. Estradiol and estriol showed a sligh but persistent fall over 24 hours prior to induction of abortion. A more rapid fall usually occurred after induction, with a consistent fall around the time of fetal death. Progesterone and HCS usually fell much less before and immediately after fetal death. A marked rise in estradiol sometimes occurred before fetal death, particularly in the intraamniotic PGE2 and urea subgroup. Estriol levels declined more rapidly before than after fetal death, whereas fetal death had less consistent effects on the other hormones. All hormones had usually fallen considerably at the time of abortion, and in some individuals marked fluctuations in hormone levels were seen.     

Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene

Chronic butylated hydroxytoluene (BHT) treatment after a single administration of a carcinogen increases lung tumor multiplicity in some inbred strains of mice. We report that BALB/cOla and BALB/cByJ mice given a low dose (10 microg/g of body weight) of 3-methylcholanthrene (MCA) develop no lung tumors unless this is followed by chronic BHT exposure. Slightly higher MCA doses (15 and 25 microg/g) induce low lung tumor multiplicities (0.6 and 1.9 tumors/mouse, respectively) that are increased 12-26-fold by chronic BHT administration. This low-dose MCA/BHT model in BALB mice will facilitate the identification of genes regulating susceptibility to lung tumor promotion and pulmonary chemopreventative agents that act at a postinitiation site.     

Cooperative thermal transitions of bovine and human apo-alpha-lactalbumins: evidence for a new intermediate state

The thermal denaturation of bovine and human apo-alpha-lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo-alpha-lactalbumin possesses a thermal transition with a midpoint about 25-30 degrees C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10-15 degrees C, while DSC shows the transition within the region of 15-20 degrees C. The non-coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311-315] and has physical properties intermediate between the native and molten globule states.