Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

MODULATION OF THE HEAT SHOCK UBIQUITIN POOL IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Immunoblotting experiments performed with an anti-ubiquitin antibody revealed that Skeletonema costatum (Grev.) Cleve cells contained free ubiquitin as well as ubiquitin conjugated to various endogenous proteins. A temperature shift from 18° to 30°C greatly increased the total amount of ubiquitin and particularly the ubiquitin fraction in high molecular mass conjugates. A solid-phase immunoassay indicated values of 0.031 ± 0.004 pmol·10^?6 cells for free ubiquitin and 0.046 ± 0.004 pmol·10^?6 cells for conjugated ubiquitin for cells grown at 18°C, and 0.056 ± 0.008pmol·10^?6cells and 0.21 ± 0.03 pmol·10^?6cells, respectively, after a temperature increase from 18° to 30°C. Cell-free extracts of S. costatum were equally able to form thiol ester linkages with ¹²⁵I-ubiquitin in an adenosine triphosphate–dependent manner at 18° C and at 30°C. Cell-free extracts were also able to conjugate ¹²⁵I-ubiquitin to endogenous proteins, but the ubiquitin conjugation rate at 30°C was lower than at 18°C. Incubation of S. costatum for 3 h at 30°C and then for 3 h at 18°C resulted in the formation of high amounts of ubiquitin conjugates, suggesting that partially inactive or denaturated proteins accumulate during heat stress. These denaturated proteins are then conjugated to ubiquitin very efficiently when the physiological temperature is restored. Thus, S. costatum cells contain ubiquitin and an active ubiquitin conjugation system responding to stress conditions (temperature stress). The intracellular concentration of ubiquitin conjugates is most likely limited by the availability of protein substrates to be conjugated rather than by ubiquitin-conjugating activity.     

Synthesis of 125I-ubiquitin conjugates in extracts of Lemna minor

Low levels of ubiquitin conjugating activity are typically detectedin the green tissues of plants, an observation that may at leastpartially explain why no method to purify multi-ubiquitinatedproteins from photosynthetic cells has been reported in theliterature. The present paper provides a contribution to improvethe available methodology for the isolation of an efficientubiquitin conjugating system from photosynthetic cells. We haveselected Lemna minor L. as a plant system and have developeda simple and rapid methodology to synthesize and purify highmolecular mass ubiquitin-protein conjugates, formed with endogenoussubstrates and exogenous ¹²⁵I-ubiquitin, using small amounts(<2g) of green tissue. It is demonstrated that L. minor possessesan ATP-dependent activity capable of forming ubiquitin conjugateswith endogen ous proteins in vitro. Anion exchange chromatographyon diethylaminoethyl-cellulose provides a simple and rapid techniqueto remove endogenous ubiquitin and to concentrate and partiallypurify the enzyme system responsible for ubiquitin conjugatingactivity. This enriched fraction has therefore been utilizedto syn thesize high molecular mass ¹²⁵I-ubiquitin conjugatesformed with L. minor proteins. These conjugates were subsequentlypurified by directly loading the reaction mixture on a SephacrylS-300 gel filtration column, with no requirement for additionalconcentration or purification steps. This methodology is highlyreproducible. Key words: Ubiquitin, ubiquitin-protein conjugates, synthesis, purification, Lemna minor     

Ubiquitin metabolism in ts85 cells, a mouse carcinoma line that contains a thermolabile ubiquitin activating enzyme

Red blood cell-mediated microinjection was used to introduce radioiodinated ubiquitin into ts85 cells, a mouse cell line that contains a thermolabile ubiquitin-activating enzyme (E1). The proportion of ubiquitin present as histone conjugates, high molecular weight conjugates, and free molecules was then determined by gel electrophoresis and autoradiography. When ts85 cells were incubated at the nonpermissive temperature, 39.5 degrees C, high molecular weight conjugates accumulated. This unexpected result was confirmed by Western blot analyses. To determine whether ubiquitin conjugates formed under nonpermissive conditions or merely persisted after the temperature increase, ts85 cells were incubated at 39.5 degrees C to generate large amounts of conjugates and then shifted to 42 degrees C. The higher temperature resulted in a 25% reduction in conjugates, but upon return to 39.5 degrees C, the ubiquitin conjugates were restored to pre-42 degrees C amounts. Since all changes in ubiquitin conjugate levels occurred above 39.5 degrees C, ts85 cells can couple ubiquitin to cellular proteins even after prolonged culture at nonpermissive temperatures. Western blot analyses showed that less than 10% of the E1 molecules present in ts85 cells at 31 degrees C remained after 2 h at 39.5 degrees C. However, when 125I-ubiquitin was added to extracts from heated ts85 cells an apparent high molecular weight form of E1 and thiol ester adducts between ubiquitin and the E2 carrier proteins were detected by electrophoresis at 4 degrees C. Considering both in vivo and in vitro demonstrations that heated ts85 cells retain the ability to conjugate ubiquitin to endogenous proteins, considerable caution must be exercised in the design and interpretation of proteolysis experiments using this mutant cell line.     

The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates

ATP, ubiquitin-dependent proteolysis proceeds through covalent intermediates between target proteins destined for degradation and the 8,600-Da polypeptide ubiquitin. The ubiquitin moiety therefore represents a sensitive immunological marker for the specificity and function of this novel post-translational modification. Methods are described for the immunochemical detection of ubiquitin conjugates immobilized on nitrocellulose filters following electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. A further modification allows quantitation of conjugated ubiquitin to the exclusion of free polypeptide. Comparisons of conjugate pools in rabbit reticulocytes and erythrocytes demonstrate that 83 +/- 3% and 31 +/- 0.2%, respectively, of total intracellular ubiquitin exists covalently bound to target proteins. Similar large proportions of conjugated ubiquitin were found in three tissue culture cell lines. Subcellular fractionation revealed that 25% of total ubiquitin conjugates of reticulocytes sediment with the 22,000 X g stromal fraction with the remainder found in the 100,000 X g supernatant. In contrast, significant levels of erythrocyte ubiquitin conjugates occur only in the 100,000 X g supernatant, suggesting ubiquitin-mediated proteolysis actively degrades stromal components lost during terminal maturation. Reticulocytes retain their full complement of active ubiquitin during maturation indicating the concomitant decline in energy-dependent proteolysis does not result from ubiquitin inactivation. That the lower level of ubiquitin conjugates and the accompanying rate of energy-dependent proteolysis in erythrocytes is a consequence of limited substrate availability is suggested by observed increases in conjugate pools and induction of specific ubiquitin-protein adducts on incubation with either phenylhydrazine or sodium nitrite.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.     

Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis

To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.     

ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle

A soluble ATP/Mg2-dependent proteolytic system from rabbit cardiac muscle has been identified (m ca. 310 kDa) and purified ca. 9-fold. This enzyme which splits the substrate [3H]globin and 125I-bovine serum albumin (125I-BSA) has many similarities to the ATP-dependent proteolytic enzyme system from reticulocytes which utilizes ubiquitin: 1) The specific activities in reticulocyte lysates and cardiac muscle extracts are of the same magnitude (0.5-1 arb. unit/mg). 2) The binding and elution behavior on DEAE-cellulose is similar. 3) In both cases the pH optimum (substrate 125I-BSA) is pH 7.6. 4) Both enzymes are inhibited by hemin, NEM and iodoacetate but not e.g. by leupeptin, or inhibitors of serine proteases. 5) Neither enzyme system can utilize ATP-analogs such as AMP-CPP, AMP-PCP, AMP-PNP or ATP-gamma-S. There are however also significant differences: 1) The enzyme system from cardiac muscle is fully active in the absence of ubiquitin and cannot be activated by this peptide. 2) The enzyme from cardiac muscle can degrade methylated BSA. 3) The cardiac muscle enzyme can be further purified on Sepharose 4B; the enzyme from reticulocytes is inactivated by this procedure. 4) The cardiac enzyme cannot be inactivated by ribonuclease as the reticulocyte counterpart. Although ubiquitin does not appear to play a role in the isolated ATP/Mg2-dependent proteolytic system from cardiac muscle, it is demonstrated for the first time that 125I-ubiquitin can be conjugated to a wide variety of cardiac muscle proteins in vitro in an ATP-dependent manner. Apparent molecular masses of major conjugates were: 185 kDa, 140 kDa, 85 kDa, 65 kDa, 46 kDa, 38 kDa and 36 kDa as estimated by discontinuous SDS gel electrophoresis. Addition of purified phosphorylase kinase to cardiac muscle extract changed the ubiquitination pattern by the appearance of two novel protein bands. It is concluded that the ATP/Mg2-dependent proteolytic system of cardiac muscle must be differentiated from the proteolytic system of reticulocytes mainly because of its ubiquitin-independence. Nevertheless the conjugation of 125I-ubiquitin to many muscle proteins is a strong indication for a crucial role of this interesting peptide in striated muscle.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.     

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.     

Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.     

Effects of aging in vitro on intracellular proteolysis in cultured rabbit lens epithelial cells in the presence and absence of serum

Summary Alterations in proteolytic capabilities have been associated with abnormalities in the aged eye lens, but in vivo tests of this hypothesis have been difficult to pursue. To simulate aging, we cultured cells from an 8-yr-old rabbit to early (population-doubling level 20 to 30) and late (population-doubling level > 125) passage. Long-lived (t_1/2>10 h) and short-lived (t_1/2<10 h) intracellular proteins were labeled with [³H]leucine, and the ability of the cells to mount a proteolytic response to the stress of serum withdrawal was determined. For early passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 62 and 39 h, respectively. For late-passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 58 and 43 h, respectively. The net increase in intracellular proteolysis in the absence of serum was 59 and 35% for early and late-passage cells, respectively. Thus, in vitro-aged rabbit lens epithelial cells mount only 60% the proteolytic response to serum removal shown in “younger” cells. The enhanced ability of early passage cells to respond to serum removal seems to involve lower homeostatic levels of proteolysis in the presence of serum and greater enhancement of proteolysis in the absence of serum. Less than 2% of the protein is in the pool of short-lived proteins. Rates of proteolysis of short-lived proteins in the presence and absence of serum were indistinguishable. With respect to basal proteolytic rates in the presence of serum and ability to mount a proteolytic response upon serum withdrawal, these rabbit lens epithelial cells are similar to bovine lens epithelial cells and fibroblasts. This work was supported in part by contract 53-3K06-5-10 U.S. Department of Agriculture, Washington, DC, Massachusetts Lions Eye Research FUnd, Inc., the Daniel and Florence Guggenheim Foundation, and a grant EY00362 from the National Eye Institute, Bethesda, MD.     

Kinetics of 125I-ubiquitin conjugation with and liberation from rabbit reticulocyte stroma

The breakdown of mitochondria-containing stroma of rabbit reticulocytes is an ATP- and ubiquitin-dependent process and there is no evidence for an ATP-dependent but ubiquitin-independent proteolysis in these cells. The ubiquitin conjugate formation with heat-denatured stroma proteins is about one-fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: an ATP-dependent and hemin-resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and a release of ubiquitin catalyzed by an isopeptidase activity.     

Ubiquitination of neuronal nitric-oxide synthase in vitro and in vivo

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of (125)I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme.     

Identification of a ubiquitin- and ATP-dependent protein degradation pathway in rat cerebral cortex.

To investigate the existence of a ubiquitin-dependent protein degradation system in the brain, the proteolytic activity of the cerebral cortex was examined. The soluble extract of rat cerebral cortex degraded 125I-radiolabeled lysozyme in an ATP-dependent manner. The ATP-dependent proteolysis was suppressed with iodoacetamide, which inhibits ubiquitin conjugation, and was abolished by blocking of the amino residues of lysozyme. These results suggest the participation of ubiquitination in the proteolytic activity. An ATP-dependent 125I-ubiquitin-conjugating activity was detected in fraction II from the cerebral cortex. The presence of ATP-dependent proteolytic activity which acted preferentially on ubiquitinated lysozyme was demonstrated, using ubiquitin-125I-lysozyme conjugates as a substrate. The proteinase had a molecular mass of 1500 kDa and displayed nucleotide dependence and sensitivity to various proteinase inhibitors similar to those of the 26S proteinase complex found in reticulocytes. Dialysis of the soluble fraction caused a decrease in the proteolytic activity of ATP-dependent and preferential for ubiquitin-lysozyme conjugates and a reciprocal increase in the ATP-independent free 125I-lysozyme-degrading activity which was scarcely detected before dialysis. The former ATP-dependent proteolytic activity may play a physiological role in the brain.     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

Demonstration of a factor in fraction I of reticulocyte lysates necessary for the steady state accumulation of ubiquitin conjugates of des-75-76-ubiquitin.

Addition of des-75-76-ubiquitin (ubiquitin lacking its two C-terminal glycine residues) to reticulocyte lysates leads to the inhibition of proteolysis and the formation of conjugates between it and native ubiquitin, as demonstrated by the incorporation of both 125I-labeled des-75-76-ubiquitin and 125I-labeled ubiquitin into these conjugates. Conjugate formation is blocked by methylation of the amino groups of des-75-76-ubiquitin, consistent with the concept that the conjugates represent attachment of the ubiquitin alpha-carboxyl group to amino groups of des-75-76-ubiquitin. The lack of significant direct competition for conjugate formation by typical ubiquitinatable proteolysis substrates or by des-73-76-ubiquitin, together with differences in conjugate formation between des-73-76-ubiquitin and des-75-76-ubiquitin demonstrated earlier, indicates that the enzyme involved recognizes the ubiquitin sequence as a substrate for ubiquitination. Increasing concentrations of native ubiquitin first increase and then reduce the steady state level of conjugates of the des-75-76-protein, the inhibitory effects of high concentrations consistent with competition by native ubiquitin for conjugate formation. Upon fractionation of reticulocyte lysates, a factor essential to the net synthesis of conjugates of des-75-76-ubiquitin was demonstrated to be present in Fraction I and to behave as a protein of molecular weight 38,000. The role in this system of a factor from Fraction I other than ubiquitin indicates that a novel pathway is involved.     

Effect of steroid hormones on sulfated oviductal glycoprotein secretion by oviductal explants in vitro

Explants of rabbit ampullary and isthmic tissue were cultured 4 days with and without exogenous steroids, and the sulfated oviductal glycoprotein (SOG) concentration in the explant culture supernatants was determined. Tissues cultured with progesterone plus estrogen secreted significantly more SOG than control tissues, whereas tissues cultured with estrogen alone did not. Ampullary tissues cultured with progesterone plus estrogen secreted significantly more SOG than control tissues on Days 2 and 3, whereas SOG secretion by isthmic tissues was significantly above control secretion on Day 4. Ampullary and isthmic tissues differed significantly in their secretory capacity. Maximum ampullary SOG secretion was approximately 650 ng SOG/mg tissue/day. Maximum isthmic SOG secretion was approximately 30 ng SOG/mg tissue/day. These findings suggest that the oviduct is composed of discrete functional regions that provide support to gametes and developing embryos through the unique secretory characteristics of each region.