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J Driscoll

Publications and source records attributed to J Driscoll.

16 recordsLinked to original sources
PubMed1992

Proteasome subunits encoded in the MHC are not generally required for the processing of peptides bound by MHC class I molecules.

Antigen processing provides major histocompatibility complex (MHC) class I molecules with short peptides, which they selectively bind and present to cytotoxic T lymphocytes. The proteolytic system generating these peptides in the cytosol is unidentified, but their delivery into the endoplasmic reticulum is mediated by the TAP1-TAP2 transporter encoded in the MHC class II region. Closely linked to TAP1 and TAP2 are genes for the LMP2 and LMP7 proteins, which resemble components of proteasomes, proteolytic complexes known to degrade cytosolic proteins. This association has led to the common assumption that proteasomes function in this immunological pathway (discussed in ref. 15). We now show that the expression of stably assembled class I molecules and apparently normal peptide processing can be completely restored in the absence of LMP2 and LMP7 in the human lymphoblastoid cell line mutant 721.174 (refs 16, 17). The identity of LMP7 is directly confirmed by reconstitution of a proteasomal subunit after gene transfer. These results therefore dispute the hypothetical involvement of proteasomes in antigen processing, although a more subtle effect of LMP2 and LMP7 cannot be ruled out.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

An ATP-stabilized inhibitor of the proteasome is a component of the 1500-kDa ubiquitin conjugate-degrading complex.

Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.

Adenosine Triphosphate↗
PubMed1991

Structural and serological similarity of MHC-linked LMP and proteasome (multicatalytic proteinase) complexes.

Major histocompatibility complex (MHC) class I molecules associate with peptides derived from endogenously synthesized antigens. Cytotoxic T-lymphocytes can thus scan class I molecules and bound peptide on the surface of cells for foreign antigenic determinants. Recent evidence demonstrates that the products of trans-acting, non-class I genes in the class II region of the MHC are required in the class I antigen-processing pathway. There are genes (called HAM1 and HAM2 in the mouse) in this region that encode proteins postulated to be involved in the transport of peptide fragments into the endoplasmic reticulum for association with newly synthesized class I molecules. But, the mechanism by which such peptide fragments are produced remains a mystery. At least two genes encoding subunits of the low-molecular mass polypeptide (LMP) complex are tightly linked to the HAM1 and HAM2 genes. We show that the LMP complex is closely related to the proteasome (multicatalytic proteinase complex), an intracellular protein complex that has multiple proteolytic activities. We speculate that the LMP complex may have a role in MHC class I antigen processing, and therefore that the MHC contains a cluster of genes required for distinct functions in the antigen processing pathway.

Animals↗
PubMed1991

A calcium-activated protease from Alzheimer's disease brain cleaves at the N-terminus of the amyloid beta-protein.

Alzheimer's disease, Down's syndrome, and to a far lesser extent, normal aged brains exhibit abnormal extracellular deposits of amyloid. The major component of brain amyloid is the beta-protein, a 4Kd fragment of the larger beta-protein precursor. The finding of the abnormally processed beta-protein and a protease inhibitor (alpha 1-antichymotrypsin) in the amyloid deposits prompted us to search for proteases which may generate the beta-protein from its precursor. We now report on the presence and partial purification of one such proteolytic activity from Alzheimer's brain. Normal physiologic C-terminal cleavage of the secreted form of the beta-protein precursor occurs in the middle of the beta-protein suggesting that the beta-protein accumulates due to an alternative degradation pathway. We propose here that the protease activity we describe participates in this abnormal pathway.

Alzheimer Disease↗
PubMed1990

The proteasome (multicatalytic protease) is a component of the 1500-kDa proteolytic complex which degrades ubiquitin-conjugated proteins.

Mammalian cells contain two large proteolytic complexes, the 650-kDa proteasome (or multicatalytic protease) and the 1500-kDa (26 S) Ubiquitin-conjugate-degrading enzyme. Since the proteasome is also required for the ATP-dependent degradation of ubiquitinated proteins, we tested whether it may be a component of the larger complex. The proteasome normally is soluble in 38% ammonium sulfate. However, after preincubation of reticulocyte extracts with ATP, several proteasome activities appeared in the 38% ammonium sulfate pellet, including the ability to degrade hydrophobic peptides and 14C-casein. Also, following preincubation with ATP, the precipitable fraction could degrade 125I-lysozyme-ubiquitin (Ub) conjugates. The activities were not present after incubation without ATP or with a nonmetabolizable ATP analog. Nondenaturing gel electrophoresis indicated the ATP-dependent appearance of a new band which degraded proteasome substrates, and reacted with an anti-proteasome monoclonal antibody on Western blot. This new band appeared larger than the proteasome and migrated similarly to the larger Ub-conjugate-degrading complex. The formation of the larger complex required factor(s) present in the 38% ammonium sulfate pellet and either the 40-80% fraction or the purified proteasome from reticulocytes or muscle. After complex formation, hydrolysis of Ub-protein conjugates and also the non-ubiquitinated substrate, casein, was stimulated severalfold by ATP, but non-metabolizable ATP analogs had little or no effect. Thus, the proteasome corresponds to component CF-3 of Ganoth et al. (Ganoth, D., Leshinisky, E., Eytan, E., and Hershkov, A. (1989) J. Biol. Chem. 263 12412-12419) and undergoes an energy-dependent association with other factors to form the 1500-kDa, ATP-requiring proteolytic complex.

Adenosine Triphosphate↗
PubMedDate not supplied

Evaluation of tympanic membrane thermometer for use with pediatric patients.

To determine if a tympanic membrane thermometer is of benefit on a pediatric unit. Temperature readings using a tympanic membrane thermometer and an electronic thermometer were compared for 295 paired observations. The electronic temperature readings were done by rectal (n = 32), oral (n = 65), and axillary (n = 198) routes. Differences in tympanic and electronic readings were compared by paired t-tests, and the readings were also correlated. The nursing time in seconds was also compared between electronic and tympanic measurements. Parents and nurses rated their opinion of each type of measurement on a 0-5 Likert scale. Nurses rated the patient's response using the same scale. Results indicate that temperature readings differ by an average of .2- .5 degree between tympanic and electronic thermometers. Correlations are statistically significant but of low-to-moderate strength. Parents and nurses did not rate the types of instruments differently. The nurses rated the electronic axillary method as being more acceptable to toddlers. The tympanic membrane measurement took an average of 30-38 seconds less time to take. Tympanic membrane thermometry saves nursing time. Although the correlations were not strong between electronic and tympanic membrane measurements, the tympanic reading was closer to the electronic rectal reading than to axillary or oral readings.

Adolescent↗