Pathological aggregation of the transactive response DNAbinding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease. TDP-43 aggregation appears to be largely driven by its low-complexity domain (LCD), which also has a high propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of TDP-43 LCD pathological aggregation and, most importantly, the relationship between the aggregation process and LLPS remains largely unknown. Here, we show that amyloid formation by the LCD is controlled by electrostatic repulsion. We also demonstrate that the liquid droplet environment strongly accelerates LCD fibrillation and that its aggregation under LLPS conditions involves several distinct events, culminating in rapid assembly of fibrillar aggregates that emanate from within mature liquid droplets. These combined results strongly suggest that LLPS may play a major role in pathological TDP-43 aggregation, contributing to pathogenesis in neurodegenerative diseases.
Oxidative stress is an important factor in the etiology and pathogenesis of diabetes. We investigated changes in mitochondrial production of reactive oxygen species (ROS) and mitochondrial antioxidant defense systems in different tissues of streptozotocin (STZ)-induced diabetic rats. Our results show that increased ROS production and oxidative stress differentially affect mitochondrial and cytosolic glutathione (GSH) metabolism. Of the four tissues investigated, the pancreas, kidney, and brain appear to be affected more severely than the liver. We show a five-to eightfold increase of cytochrome P450 2E1 (CYP2E1) and glutathione Stransferase (GST) A4-4 levels in mitochondria from STZ-treated rat tissues compared with those in nondiabetic rat tissues, suggesting possible roles in the disease process. Transient transfection of COS cells with CYP2E1 cDNA caused a similar accumulation of CYP2E1 and GST A4-4 in mitochondria and increased production of mitochondrial ROS. Our results also show an increase in steady-state levels of Hsp70 in the mitochondrial and cytosolic fractions of different tissues of diabetic rats. These results indicate, for the first time, a marked increase in mitochondrial oxidative stress in target tissues of STZ-treated rats and implicate a direct role for mitochondrial CYP2E1 in the generation of intramitochondrial ROS.
We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O 2 for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O 2 species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nM H89 or 50 nM myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions. Cytochrome c oxidase (CcO) 3 is the terminal oxidase of the mitochondrial electron transport chain, whose activity is modulated in response to O 2 tension and the work load of the tissue (1-6). This rate-limiting enzyme is an important site of regulation of mitochondrial respiration and oxidative phosphorylation (7). In the yeast, altered CcO activity in response to aerobic and anaerobic conditions is associated with the differential expression of the two isologs of the CcO Vb gene (8), although the precise mechanism by which the mammalian CcO modulates its activity remains unknown. Mitochondrial electron transport chain complexes are major sources of cellular ROS under both normoxic and hypoxic conditions (9, 10). Hypoxia-tolerant and hypoxia-sensitive human glioma cells exhibit distinct patterns of mitochondrial function in response to hypoxia (9, 11). Submitochondrial particles exposed to hypoxic conditions in vitro show reduced CcO activity (1,10,12). Some studies also suggest that the myocardial ischemia/reperfusion injury is manifested through altered CcO activity and reduced mitochondrial oxidative phosphorylation (13,14).Protein kinases have been suggested to play a role in the modulation of myocardial ischemia/reperfusion injury (15), although the roles of different cellular components in mediating this injury remain unclear. The presence of PKA and PKC activities in the mitochondrial inner membrane-matrix compartment and the role of PKC-mediated phosphorylation in the regulation of pyruvate dehydrogenase activity are well established (16). An 18-kDa subunit of the NADH dehydrogenase (complex I) (17)...
Glutathione (GSH) conjugating enzymes, glutathione S-transferases (GSTs) are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus and plasma membrane. The regulation and function of GSTs have implications in cell growth, oxidative stress, as well as in disease progression and prevention. Of the several mitochondria localized forms, GSTK (GST kappa) is mitochondria-specific since it contains N-terminal canonical and cleavable mitochondria targeting signal. Other forms, like GST alpha, mu and pi purified from mitochondria are similar to the cytosolic molecular forms or “echoproteins”. Altered GST expression has been implicated in hepatic, cardiac and neurological diseases. Mitochondria-specific GSTK has also been implicated in obesity, diabetes and related metabolic disorders. Studies have shown that silencing the GSTA4 (GST alpha) gene resulted in mitochondrial dysfunction, as was also seen in GSTA4 null mice which could contribute to insulin resistance in type 2 diabetes. This review highlights the significance of mitochondrial GST pool, particularly the mechanism and significance of dual targeting of GSTA4-4 under in vitro and in vivo conditions. GSTA4-4 is targeted in the mitochondria by activation of the internal cryptic signal present at the C-terminus of the protein by protein kinase-dependent phosphorylation and cytosolic heat shock protein (Hsp70) chaperon. Mitochondrial GSTpi, on the other hand, has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N-terminal region. Both physiological and pathophysiological implications of GST translocation to mitochondria have been discussed in this review.
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