Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclearencoded 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 (e") 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 @' ) 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 @ 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.Keywords: molecular chaperone ; mitochondria; stress response; heat shock ; gene activation. Molecular chaperones are found in all of the major sub-celMar compartments and have been shown to play essential functions in the folding and subcellular targeting of proteins. In addition, the ability of cells to survive stresses such as elevated temperatures, exposure to heavy metals and amino acid analogues is dependent on the induction of molecular chaperones (re- chaperones are essential for cell viability [lo, 15-17]. The upregulation of mitochondrial chaperones in response to metabolic insults such as heat shock, glucose deprivation, amino acid analogues and agents that impair energy metabolism [ 18 -191, suggest that they also play an important role in maintaining homeostasis in mitochondria when a general stress is applied to the cell. However, it is not known if mitochondrial chaperones are induced specifically in response to the application of a selective stress to mitochondria.Chaperone synthesis can be regulated in part by modulation of the message stability and the frequency of translation initiation, but is regulated mainly through specific transcriptional factors termed heat-shock factors (reviewed in [20]). The binding of heat-shock factors to DNA regulatory sequences, called heat-shock elements, promotes the expression of heat-shock genes, and is thought to be modulated by a number of processes such as oligomerisation, phosphorylation and i...

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.

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.

The focus of this review is to summarise the known relationships between the expression of heat shock protein 60 (Hsp60) and its association with the pathogenesis of Type 1 and Type 2 diabetes mellitus. Hsp60 is a mitochondrial stress protein that is induced by mitochondrial impairment. It is known to be secreted from a number of cell types and circulating levels have been documented in both Types 1 and 2 diabetes mellitus patients. The biological significance of extracellular Hsp60, however, remains to be established. We will examine the links between Hsp60 and cellular anti- and proinflammatory processes and specifically address how Hsp60 appears to affect immune inflammation by at least two different mechanisms: as a ligand for innate immune receptors and as an antigen recognised by adaptive immune receptors. We will also look at the role of Hsp60 during immune cell activation in atherosclerosis, a significant risk factor during the pathogenesis of diabetes mellitus.

Diabetes mellitus is the most common metabolic disorder characterized by hyperglycemia and associated malfunctions of the metabolism of carbohydrates, proteins, and lipids. There is increasing evidence of a relationship between diabetes and vascular dementia. Interestingly, hyperglycemia-linked neuroinflammation in the central nervous system is considered to play a key role during vascular dementia in diabetic patients. However, the mechanisms responsible for the relationship between hyperglycemia and neuroinflammation is not clearly understood. Diabetes-induced alternations in the blood-brain barrier permit high glucose influx into the brain cells via glucose transporters and promote oxidative stress through overproduction of reactive oxygen species. Despite many studies demonstrating a link between oxidative stress and mitochondrial dysfunction, the relationship between mitochondrial dysfunction and neuron inflammation during hyperglycemia remains to be established. In this review, we will focus on diabetes-induced changes in the central nervous system and the role of mitochondrial heat shock protein 60 (HSP60) as an initiator of oxidative stress and potential modulator of neuroinflammation. We suggest that oxidative stress-mediated mitochondrial dysfunction stimulates the upregulation of mitochondrial heat shock protein 60 (HSP60) and ultimately initiates inflammatory pathways by activating pattern recognition receptors. HSP60 also could be a focal point in the development of a biomarker of neuroinflammation as HSP60 is known to be significantly elevated in diabetic patients. Interestingly, extracellular secretion of HSP60 via exosomes suggests that inflammation could spread to neighboring astrocytes by activating pattern recognition receptors of astrocytes via neuronal exosomes containing HSP60. A mechanism for linking neuron and astrocyte inflammation will provide new therapeutic approaches to modulate neuroinflammation and therefore potentially ameliorate the cognitive impairment in diabetic brains associated with vascular dementia.

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.

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.

There is increasing evidence that mitochondrial dysfunction and oxidative stress may be integral to the pathogenesis of type 2 diabetes mellitus. Heat shock protein (Hsp60) is a mitochondrial stress protein known to be induced under conditions of mitochondrial impairment. Although this intracellular protein is normally found in the mitochondrion, several studies have shown that this protein is also present in systemic circulation. In this study, we report the presence of elevated levels of Hsp60 in both saliva and serum of type 2 diabetic patients compared to non-diabetic controls. Hsp60 was detectable in the saliva of 10% of control and 93% of type 2 diabetic patients. Levels detected were in the range of 3-7 ng/ml in control and 3-75 ng/ml in type 2 diabetic patients. Serum Hsp60 levels in the range of 3-88 ng/ml were detected in 33% of control subjects, and levels in the range of 28-1,043 ng/ml were detected in 100% of type 2 diabetic patients. This is the first reporting of the presence of mitochondrial stress protein in salivary secretions. The serum Hsp60 levels were 16-fold higher compared to those in saliva, and there was a good positive correlation between salivary and serum Hsp60 levels (r=0.55). While the exact mechanisms responsible for the secretion of Hsp60 into biological fluids such as saliva and blood are not yet known. The presence of this molecular marker of mitochondrial stress in saliva offers a non-invasive route to further investigate the biological functions of extracellular Hsp60 in type 2 diabetes mellitus and other conditions.