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J F Dice

Publications and source records attributed to J F Dice.

At least 37 records · Page 2Linked to original sources
PubMed1995

Batten disease fibroblasts in culture accumulate mitochondrial ATP synthase subunit 9.

Batten Disease is a lysosomal storage disease in which the major component that accumulates is subunit 9 of mitochondrial ATP synthase. Whether or not fibroblasts in culture exhibit this phenotype is controversial. We show that fibroblasts from a human Batten Disease patient and from a mouse model of this disease exhibit autofluorescent inclusion bodies. We also demonstrate that levels of ATP synthase subunit 9 are elevated in these diseased fibroblasts when compared to control cells. However, the exact growth state of the human fibroblasts was critical, and this factor probably accounts for discrepencies in the literature.

Animals↗
PubMed1995

Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation.

Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.

Animals↗
PubMed1994

Selective binding and uptake of ribonuclease A and glyceraldehyde-3-phosphate dehydrogenase by isolated rat liver lysosomes.

Ribonuclease A (RNase A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are selectively taken up and degraded by isolated rat liver lysosomes by very similar processes. The uptake and degradation of both of these proteins are stimulated by the heat shock cognate protein of 73 kDa and ATP/Mg2+. Both binding and uptake of RNase A and GAPDH by lysosomes are saturable, and uptake of RNase A and GAPDH requires a protease-sensitive component within the lysosomal membrane. GAPDH competes for binding and uptake of RNase A by lysosomes and vice versa while another protein, ovalbumin, does not compete. RNase S-peptide (amino acids 1-20 of RNase A) also competes for RNase A binding and uptake by lysosomes, while RNase S-protein (amino acids 21-124 of RNase A) does not compete. The uptake of RNase A by lysosomes appears to involve an intermediate step in which approximately 2 kDa of the polypeptide's COOH terminus remains outside lysosomes while the remainder is inside the lysosomal lumen.

Amino Acid Sequence↗
PubMed1994

Two related proteolipids and dolichol-linked oligosaccharides accumulate in motor neuron degeneration mice (mnd/mnd), a model for neuronal ceroid lipofuscinosis.

In this study, we show that two biochemical markers of neuronal ceroid lipofuscinoses (NCLs) are present in a mutant mouse (mnd/mnd) that exhibits symptoms of the disease. Subunit c of the mitochondrial F1F0-ATP synthase, a proteolipid that accumulates in storage bodies of most forms of NCL and several animal models, is dramatically increased in mnd/mnd mouse brain, kidney, liver, heart, and pancreas. Interestingly, another related proteolipid, subunit c of the vacuolar H(+)-ATPase, also accumulates in several mnd/mnd tissues. The molar ratio of the vacuolar subunit c to the F1F0 subunit c is approximately one to two in enriched storage bodies from brain. The relative accumulation of the vacuolar subunit c correlates with its abundance in normal tissues. It appears in decreasing amounts in brain, kidney, and liver and is not detected in heart or pancreas. Aged mice and two mutant mouse lines, juvenile bare (jb) and mucopolysaccharidosis, type VII (gusmps), did not accumulate either of these proteolipids. Dolichol-linked oligosaccharides also accumulate in NCLs and are increased 17-fold in mnd/mnd mouse brain. Thus, mnd/mnd mice seem to be an excellent model for NCLs since they not only share clinical signs and histopathology, but also two biochemical markers. The accumulation of the vacuolar subunit c in this model may prove to be a marker for distinguishing different forms of NCLs.

Amino Acid Sequence↗
PubMed1993

Selective release of peptides from lysosomes.

We demonstrate a selective release of peptides by lysosomes in vitro. A lysosomal fraction from human fibroblasts that had previously endocytosed [3H]ribonuclease A was incubated for 2 h, and radioactivity released into the medium and radioactivity retained within lysosomes were analyzed. A variety of radiolabeled molecules including peptides of an appropriate size to serve as antigens for T cell-mediated immunity were released. One small peptide was predominantly released, while others, as well as intact ribonuclease A, were predominantly retained. A 4-5-fold range was also evident in the relative release of three 3H-labeled tripeptide probes of similar charge derived from the sequence of ribonuclease A. This selectivity and the fact that similar peptide degradation fragments were also released and retained by intact cells after endocytosis of [3H]ribonuclease A argues strongly that the release observed in vitro is physiological and not due to damaged lysosomal membranes.

Cells, Cultured↗
PubMed1993

Polypeptide import and degradation by isolated lysosomes.

We reported previously that lysosomes derived from human diploid fibroblasts import and degrade polypeptides and that these processes are stimulated by ATP and by the heat shock cognate protein of 73 kDa (hsc73). We now report several new aspects of this in vitro proteolytic pathway. (a) Among four polypeptides tested, this pathway appears to be selective for those containing KFERQ-like peptide motifs. (b) Substrate proteins specifically bind to a protein-containing site on lysosomal membranes. (c) Lysosomes derived from serum-deprived cells are twice as active as those from serum-supplemented cells. (d) A portion of intracellular hsc73 is associated with lysosomes, and the amount of lysosomal hsc73 increases in response to serum withdrawal. Additional characterization of this proteolytic pathway not reported previously shows that intact lysosomes are required, the import process is saturable with an apparent Km of 5 microM for RNase S-peptide, and reducing agents activate this lysosomal import and degradation pathway.

Amino Acid Sequence↗
PubMed1992

Selective degradation of cytosolic proteins by lysosomes.

Lysosomes are able to internalize cellular proteins in a variety of ways. One pathway is selective for cytosolic proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent monolayers of cultured cells in response to deprivation of serum growth factors and applies to approximately 30% of cytosolic proteins. We have reconstituted this lysosomal degradation pathway in vitro. Uptake and/or degradation is stimulated by ATP and a member of the heat shock 70-kilodalton protein family, the 73-kilodalton constitutive heat shock protein. Several possible mechanisms of selective protein transport into lysosomes and the possible relevance of this proteolytic pathway to the processing of the amyloid precursor protein are discussed.

Alzheimer Disease↗
PubMed1992

Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein.

Lysosomal degradation of intracellular proteins during serum withdrawal is stimulated by a member of the 70-kDa heat shock protein (hsp70) family (Chiang, H.-L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989) Science 246, 382-385). This hsp70, isolated by affinity chromatography with RNase S-peptide-Sepharose, is referred to as the 73-kDa peptide recognition protein (prp73). We now report that prp73 binds to several proteins and peptides whose degradative rates are increased during serum withdrawal. prp73 also binds to the pentapeptide, KFERQ, and more weakly to most modified RNase S-peptide derivatives with a single amino acid substitution within the KFERQ sequence. Taken together, these results suggest that prp73 binds to a variety of proteins at peptide regions biochemically related to KFERQ. Three lines of evidence indicate that prp73 is the heat shock cognate protein of 73 kDa (hsc73): (a) among five hsp70s tested, hsc73 binds to RNase S-peptide most avidly, (b) both prp73 and hsc73 also bind to RNase A and aspartate aminotransferase but not to ovalbumin, lysozyme, or ubiquitin, and (c) both prp73 and hsc73 promote uptake and degradation of [3H] RNase S-peptide by lysosomes in vitro, while three other hsp70s are without activity in this assay.

Amino Acid Sequence↗
PubMed1991

Evidence for isopentenyladenine modification on a cell cycle-regulated protein.

We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.

Adenine↗
PubMed1991

Proteins containing peptide sequences related to Lys-Phe-Glu-Arg-Gln are selectively depleted in liver and heart, but not skeletal muscle, of fasted rats.

In response to serum withdrawal, when overall rates of proteolysis increase in cultured fibroblasts, proteins containing peptide regions similar to Lys-Phe-Gln-Arg-Gln (KFERQ) are targeted to lysosomes for degradation, and the intracellular concentrations of these proteins decline [Chiang & Dice (1988) J. Biol. Chem. 263, 6797-6805]. To test whether such proteins are also selectively depleted in mammalian tissues in vivo, we have used affinity-purified polyclonal antibodies to KFERQ to detect proteins containing such sequences in tissues of fed and fasted rats. Immunoreactive cytosolic proteins were partially depleted from liver and heart of fasted rats, but the time course differed for these two tissues. Immunoreactive proteins in liver were lost during days 2 and 3 of fasting, whereas such proteins in heart were depleted within day 1 of fasting. In the same fasted rats, levels of immunoreactive cytosolic proteins did not change in two skeletal muscles, the dark soleus and the pale extensor digitorum longus. Immunoreactive proteins in a myofibrillar fraction were also partially depleted in heart, but not in skeletal muscles, of fasted rats. The most likely explanation for these results is that the protein loss in different tissues upon fasting results from selective activation of different proteolytic pathways. The increased proteolysis in liver and heart of fasted animals includes activation of the KFERQ-selective lysosomal pathway, whereas increased proteolysis in skeletal muscle does not.

Amino Acid Sequence↗
PubMed1991

Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes.

We compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long-lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin-like growth factor-2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF-2 inhibited protein breakdown at concentrations well within the range of total IGF-2 known to be present in the serum of fetal and neonatal rats. The breakdown of short-lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half-life of myosin was 27 hours, and myosin breakdown was not altered by serum withdrawal applies to certain proteins, but not to others.

Animals↗
PubMed1991

Targeting specific proteins for lysosomal proteolysis.

A class of cytosolic proteins has been identified that are degraded faster (have shorter half-lives) in human diploid fibroblasts deprived of serum. In RNase A, a model protein used for these studies, a pentapeptide comprising amino acids 7-11, Lys-Phe-Glu-Arg-Gln or KFERQ, is responsible for its enhanced degradation. The cytosolic proteins that are degraded faster during serum deprivation are recognized by an antiKFERQ antibody and, therefore, probably contain variations of the KFERQ motif. These cytosolic proteins are degraded in lysosomes. Transport into lysosomes in vitro is stimulated by ATP and the heat shock cognate protein of 73 kDa (hsc73).

Adenosine Triphosphate↗
PubMed1991

A pathway of lysosomal proteolysis mediated by the 73-kilodalton heat shock cognate protein.

Cultured IMR-90 diploid human lung fibroblasts respond to withdrawal of serum or growth factors by increasing protein degradation. This increase, due to enhanced transfer of proteins into lysosomes, is specific for a class of intracellular proteins containing peptide sequences biochemically related to Lysine-Phenylalanine-Glutamate-Arginine-Glutamine (KFERQ). This peptide motif is recognized by an intracellular protein which facilitates its transfer into lysosomes in vitro and presumably, in vivo. We called this protein the peptide recognition protein of 73-kilodaltons (prp73). We have shown prp73 to be the constitutive member of the heat shock 70kD family (hsc73) by a variety of criteria. Furthermore, our reconstitution of this pathway of lysosomal degradation in vitro has provided insight in to the molecular mechanisms and requisite biochemical components.

Amino Acid Sequence↗
PubMed1990

Peptide sequences that target cytosolic proteins for lysosomal proteolysis.

Lysosomes take up and degrade intracellular proteins in cultured cells in response to serum deprivation, and in tissues of organisms in response to starvation. One mechanism by which proteins enter lysosomes for subsequent degradation requires that substrate proteins contain peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ).

Amino Acid Sequence↗
PubMed1990

Targeting of cytosolic proteins to lysosomes for degradation.

We review evidence for a pathway by which specific cytosolic proteins are targeted to lysosomes for degradation in cultured cells in response to serum withdrawal. This pathway is also activated by starvation in several rat tissues. The enhanced degradation is specific for a class of intracellular proteins containing peptide sequences related to residues 7 to 11 of ribonuclease A (RNase A). The amino acid sequence of this pentapeptide is lysine-phenylalanine-glutamate-arginine-glutamine, or, in single letter amino acid abbreviations, KFERQ. A heat shock protein of 73 kDa binds to such peptide regions in proteins and somehow mediates their transfer to lysosomes for degradation. The recent reconstitution of this lysosomal pathway of proteolysis in vitro should permit detailed mechanistic analysis of how proteins are directed to and translocated across lysosomal membranes.

Animals↗
PubMed1990

A selective pathway for degradation of cytosolic proteins by lysosomes.

A lysosomal pathway of proteolysis is selective for cellular proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent cultured cells that are deprived of serum growth factors and in certain tissues of fasted animals. We have reconstituted this lysosomal degradation pathway in vitro. Transport into lysosomes requires a KFERQ-like sequence in the substrate protein and uptake and/or degradation is stimulated by ATP. A member of the heat shock 70 kDa protein family, the 73 kDa constitutive heat shock protein, binds to KFERQ-like peptide regions within proteins and, in some as yet unidentified manner, facilitates transfer of the proteins into lysosomes. Several possible mechanisms of selective protein transport into lysosomes are discussed.

Amino Acid Sequence↗