Upregulation of P-glycoprotein in rat hepatoma rho(o) cells: implications for drug-DNA interactions.

Rat hepatoma cells lacking mitochondrial DNA (rho(o) cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin). The rho(o) cells were 45-fold less sensitive to Adriamycin than the parental rho+ cells containing mitochondrial DNA. Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the rho(o) cells. This was indicated by confocal microscopy where rho+ cells exhibited thirteenfold higher cellular levels of Adriamycin than rho(o) cells. Upregulation (tenfold) of P-glycoprotein in rho(o) cells was also confirmed by Northern dot blot analysis. Since the MDR phenotype is present in rho(o) cells and upregulation of P-glycoprotein is maintained in these cells, rho(o) cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution.

Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome.

Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclear-encoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (rho 0) rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in rho 0 cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the rho 0 state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles.

Role of chaperones in the biogenesis and maintenance of the mitochondrion.

All cells depend on correctly folded proteins for optimal function. A central question in cellular biology is how such folded structures are formed and maintained, a process that is now recognized to rely heavily on a group of proteins called molecular chaperones. Molecular chaperones constitute distinct families of proteins that are ubiquitous and highly conserved from bacteria to humans. They appear to bind nonnative conformations of most, if not all, proteins, thereby preventing their aggregation and subsequent inactivation. The chaperones not only protect newly synthesized proteins during transport and folding, but also serve to maintain the cell in a healthy state during exposure to a multitude of stress conditions. Accordingly, chaperones are expressed constitutively, but their synthesis is further enhanced during stress conditions. Detailed insights into the role of molecular chaperones have come from studies of mitochondrial protein biogenesis, a process in which chaperones act as unfoldases, pulling devices, and foldases. In this review we summarize these developments and further discuss the potential role of chaperones in mitochondrial DNA metabolism and human mitochondrial disease states.

Growth of rho 0 human Namalwa cells lacking oxidative phosphorylation can be sustained by redox compounds potassium ferricyanide or coenzyme Q10 putatively acting through the plasma membrane oxidase.

Pyruvate is conventionally used as a key growth supplement for mammalian rho 0 cells that lack mitochondrial DNA and are thereby devoid of oxidative phosphorylation. We have tested the proposition that cultured rho 0 human cells can be grown using redox compounds other than pyruvate. The results show that potassium ferricyanide and coenzyme Q10 can each be used to replace pyruvate to support the growth of rho 0 Namalwa cells (a lymphoblastoid cell line). Ferricyanide and coenzyme Q10 have both been reported as substrates for a plasma membrane NADH oxidase system which is capable of re-oxidising cytosolic NADH to NAD+. These compounds are also known to stimulate the activity of this enzyme system. We interpret our data to indicate that redox support for growth of rho 0 human cells can be achieved by external electron acceptors such as ferricyanide (a plasma membrane impermeant compound), or coenzyme Q10 (an integral component of the plasma membrane oxidase), through the enhanced conversion of cytosolic NADH to NAD+. This re-oxidation of NADH enables glycolysis to function efficiently as the sole source of cellular ATP, in the absence of mitochondrial oxidative phosphorylation in rho 0 cells. This has important implications for the development of new strategies for the amelioration of the bioenergy decline that occurs in mitochondrial disease and during the human ageing process.

Sheep and other animals with ceroid-lipofuscinoses: their relevance to Batten disease.

Distinct pathological and histopathological changes distinguish the ceroid-lipofuscinoses from other storage diseases of humans and animals. These various disease entities likely reflect a variety of mutations of the same gene, or mutations of different genes associated with metabolism of the same or similar substrates. The disease in sheep most closely resembles the juvenile human disease. In it 50% of the lipopigment consists of subunit c of mitochondrial ATP synthase while the remaining constituents are considered normal for a lysosomal derived cytosome. The same subunit c has been shown to be also stored in affected English Setter, Border Collie, and Tibetan Terrier dogs, the Devon cow, and in the late infantile and juvenile human forms of disease but not in the infantile form. Thus it gives a chemical unity to at least some members of the group and allows a major conceptual change in regard to further directions of research.

Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease).

The ceroid-lipofuscinoses (Batten disease) are neurodegenerative inherited lysosomal storage diseases of children and animals. A common finding is the occurrence of fluorescent storage bodies (lipopigment) in cells. These have been isolated from tissues of affected sheep. Direct protein sequencing established that the major component is identical to the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mitochondrial ATP synthase and that this protein accounts for at least 50% of the storage body mass. No other mitochondrial components are stored. Direct sequencing of storage bodies isolated from tissues of children with juvenile and late infantile ceroid-lipofuscinosis established that they also contain large amounts of complete and normal subunit c. It is also stored in the disease in cattle and dogs but is not present in storage bodies from the human infantile form. Subunit c is normally found as part of the mitochondrial ATP synthase complex and accounts for 2-4% of the inner mitochondrial membrane protein. Mitochondria from affected sheep contain normal amounts of this protein. The P1 and P2 genes that code for it are normal as are mRNA levels. Oxidative phosphorylation is also normal. These findings suggest that ovine ceroid-lipofuscinosis is caused by a specific failure in the degradation of subunit c after its normal inclusion into mitochondria, and its consequent abnormal accumulation in lysosomes. This implies a unique pathway for subunit c degradation. It is probable that the human late infantile and juvenile diseases and the disease in cattle and dogs involve lesions in the same pathway.

Bovine ceroid-lipofuscinosis (Batten's disease): the major component stored is the DCCD-reactive proteolipid, subunit C, of mitochondrial ATP synthase.

The ceroid-lipofuscinoses (Batten's disease) are a group of recessively inherited lysosomal storage diseases of children and animals in which there is intracellular accumulation of a fluorescent lipopigment in a wide variety of cells. Lipopigment bodies isolated from pancreas, liver, kidney and brain tissue from a heifer affected with ceroid-lipofuscinosis contained between 55 and 62% protein. A dominant component comigrated on LDS-PAGE with the major low molecular weight protein stored in ovine ceroid-lipofuscinosis. It was identified by amino acid sequence and mass spectroscopy as the full subunit c of mitochondrial ATP synthase, normally found only in the inner mitochondrial membrane, where it is estimated to account for 2-4% of the membrane protein. In pancreatic lipopigment it accounted for at least 40% of the total lipopigment mass and this storage was considered specific to the disease. No other mitochondrial proteins were found in storage bodies. These results are similar to those found in studies on the ovine and the late infantile and juvenile human forms of the disease. It is concluded that bovine ceroid-lipofuscinosis is also a proteolipid proteinosis in which subunit c of mitochondrial ATP synthase is specifically stored in lysosome derived organelles.

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.

Ovine ceroid-lipofuscinosis is a proteolipid proteinosis.

The pathogenesis of the ceroid-lipofuscinoses, inherited storage diseases of children, was studied in an ovine model. This was shown to have clinical and pathological features most in common with the late infantile and juvenile human forms of the disease. The ability to study sequential changes allowed the retinal lesions to be described as a dystrophy of photoreceptor outer segments which preceded loss of the photoreceptor cells. An early decrease in amplitude of the c-wave electroretinograph was attributed to a decrease in the transpigment epithelial component. The decreased a- and b-wave amplitudes were attributed to the changes in and loss of, photoreceptor cells. The chemical components of isolated storage cytosomes were analyzed and shown to consist mostly of protein. Sequence analysis of the dominantly stored protein showed that it was identical to the DCCD reactive proteolipid or subunit c of mitochondrial adenosine triphosphate synthase and that it comprised approximately 50% of storage material. Based on the adage that the dominantly stored species should reflect the underlying biochemical anomaly, it was concluded that it was of pathogenic significance. This highly hydrophobic protein tends to extract with lipids in chloroform/methanol and is thus known as a proteolipid. Some of the remainder of the stored proteins also had this characteristic. It was concluded that ovine ceroid-lipofuscinosis was a proteinosis, more specifically a proteolipid proteinosis and as such it forms the prototype of a new class of storage diseases. Recognition of the nature of the dominantly stored chemical species has helped understanding of a variety of chemical and physical characteristics attributed to the whole pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence.

Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.

Lysosomal storage of the DCCD reactive proteolipid subunit of mitochondrial ATP synthase in human and ovine ceroid lipofuscinoses.

The ceroid lipofuscinoses (Batten's disease) are a group of neuro-degenerative lysosomal storage diseases of children and animals that are recessively inherited. In the diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. The material stored in the cells of sheep affected with ceroid lipofuscinosis is two-thirds protein. The stored material does not arise from lipid peroxidation or a defect in lipid metabolism, and the lipid content is consistent with a lysosomal origin for the storage bodies. The major protein stains poorly with Coomassie blue dye and is soluble in organic solvents. It has an apparent molecular weight of 3,500 and its amino acids sequence is identical to that of the dicyclohexylcarbodiimide (DCCD) reactive proteolipid, subunit c, of mammalian mitochondrial ATP synthases. Apart from removal of mitochondrial import sequences, it has not been modified post-translationally. At least 50% of the mass of the storage bodies is composed of this protein. A minor protein sequence related to the 17-kDa subunit of vacuolar H(+)-ATPase is also found in storage bodies isolated from pancreas. As in humans and cattle, the ovine protein is the product of two expressed genes named P1 and P2. In normal and diseased animals there are no differences in sequences between P1 cDNAs or P2 cDNAs, nor do levels of mRNAs in liver for P1 or P2 differ substantially between normal and diseased animals. Both normal and diseased sheep also express a spliced pseudogene encoding amino acids 1 to 31 of the mitochondrial import presequence. The peptides they encode differ by one amino acid; arginine-23 is changed to glutamine in the diseased sheep. Storage bodies isolated from brains and pancreas of children affected with the juvenile and late infantile forms of ceroid lipofuscinosis also contain large amounts of material that is identical to subunit c of ATP synthase. However, the protein is not present in storage bodies isolated from brains of patients affected with the infantile form of the disease, and these storage bodies contain other unidentified proteins. It is possible that the cause of ovine, juvenile and late infantile ceroid lipofuscinoses is related to a defect in degradation of the subunit c of mitochondrial ATP synthase.

Ovine ceroid-lipofuscinosis. I: Lipopigment composition is indicative of a lysosomal proteinosis.

The ceroid-lipofuscinoses are inherited lysosomal storage diseases of children and animals characterised by a fluorescent lipopigment stored in a variety of tissues. Defects in lipid metabolism or the control of lipid peroxidation have been postulated to explain their pathogenesis but the underlying biochemical defect is still unknown. In the present study lipopigment was isolated from liver, kidney, pancreas and brain of sheep affected with ceroid-lipofuscinosis. Approximately two-thirds of the lipopigment mass was protein. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed a major polypeptide band of Mr 14,800, heterogeneous polypeptides between 5,000-9,000 Mr and a major band of Mr 3,500. These were not normal lysosomal proteins. I125 radiolabeling studies indicated that they were 47% of the pancreatic lipopigment mass, the 3,500 Mr polypeptides alone accounting for 26%. Lipopigment polypeptides were not subunits of a larger protein held together by disulphide bonds. The presence of the 3,500 Mr proteins in whole affected tissue homogenates distinguished them from homogenates of normal tissues. Lipopigment phospholipids were the same species as normal lysosomal phospholipids, including bis (monoacylglycero) phosphate, a lysosomal marker. Similarly the neutral lipids, notably dolichol, ubiquinone and dolichyl esters were typical of those in lysosomal membranes. Lipopigments contained 1-1.7% metals. Analyses of them indicated a functional lysosomal origin for the lipopigment. It was concluded that low Mr proteins are specifically stored in ovine ceroid-lipofuscinosis and that this disease is a lysosomal proteinosis.