**5. Some other factors affecting VDAC conductance**

formed, glucose concentration, metabolic conditions, and the presence of tubulin-like effectors increasing VDAC voltage sensitivity [4]. Prevention of ATP production together with higher hexokinase activity favors glycolysis. Research has shown that cancer cells respire in the absence of glucose, suggesting that VDAC blocking is not absolute, and the respiration increases with the addition of glucose. Increase in respiration is attributed to the formation of ADP in the hexokinase-catalyzed reaction and subsequent stimulation of oxidative phosphorylation. Since not all VDACs are closed, preferential access to mitochondrial ATP may be allowed for the hexokinase reaction [8]. Another principle applies if ATP from cytosol is readily available for hexokinase, leading to so-called turbo effect of uncontrolled glycolysis

The product hexokinase-catalyzed reaction, glucose-6-phosphate, was found to potentiate ATP release from mitochondria with the recovery of normal metabolism, and substrate replacement, even increased by induction of release of hexokinase and glucokinase from bound to VDAC [4, 41, 50]. Therefore, the effect of hexokinase dissociation from VDAC is the subject of many studies dealing with the development of potent chemotherapy of cancer diseases.

Most interestingly, there is a view of influencing metabolism of the mitochondria by blocking VDAC after binding of glucokinase in pancreatic beta cells. The glucokinase acts as a glucose sensor to maintain glucose homeostasis also in neurons, pituitary, and endocrine K and L cells [51], as its *K*m of approximately 5 mM matches the set point of blood glucose [52]. An alternatively spliced glucokinase with an additional N-terminal sequence that promotes glucokinase binding to both mitochondria and secretory granules is expressed in pancreatic beta cells [53]. Glucose-6-phosphate formed after glucose enters beta cells acts to dissociate glucokinase from mitochondria and open VDAC permitting mitochondrial uptake of ADP, Pi and respiratory substrates, formation of ATP, and release of ATP into the cytosol. Increased cytosolic ATP/ADP then inhibits KATP channels, which causes plasmalemmal depolarization, activation of Ca2+ channels, and Ca2+-dependent exocytosis of insulin granules [8]. Then, respiratory substrates that require the passage through VDAC, such as pyruvate, lactate, and long-chain fatty acids, do not trigger insulin synthesis directly. But their permeable substrates without the need for VDAC transport such as short-chain fatty acids or methylpyruvate are capable of insulin synthesis induction [54]. Ahmed et al. [55] found that under glucotoxic conditions upregulation of VDAC1 expression occurs initiating a mitochondrial death cascade and beta cell dysfunction.

Two creatine kinase (CK) isoforms are expressed in vertebrate tissues, namely dimeric cytosolic and octameric mitochondrial located in the peripheral intermembrane space and the cristae MIM (MtCK) [56]. In addition, there are tissue-specific mitochondrial isoforms in the sarcomers of striated muscles (sMtCK), and in most other tissues, they are present as ubiquitous MtCK (uMtCK) [57]. Similar to hexokinase, creatine kinase acts as energy sensor and mediates antiapoptotic effect through VDAC-ANT complexes with preferential use of mito-

Isoenzymes are associated to subcellular structures, forming microcompartments that facilitate a functional coupling, e.g., a direct exchange of ADP and ATP between the association partners

activation and development of metabolic burst [4].

256 Mitochondrial Diseases

**4.2. Creatine kinases**

chondrial ATP [41, 58].

Besides the aforementioned intermediate filament proteins and mitochondrial kinases, the conductivity of VDAC as described by Lemasters et al. [67] is regulated by a number of other factors, for example, Bcl-2 family members, protein kinase A, glycogen synthase 3β, protein C kinase ε, NADH, Ca2+, ATP, and glutamate [68].

Protein kinase A phosphorylates VDAC and thus increases its sensitivity to tubulin and decreases VDAC conductance [69]. Glycogen synthase 3β–mediated VDAC phosphorylation promotes VDAC opening [70].

The Bcl-2 protein family belongs to the key factors in the regulation of apoptosis , modulation of Ca2+ and signal transduction pathway. The Bcl-2 protein as anti-apoptotic protein prevents the release of cytochrome c and the activity of caspase. Bax, the main regulator of Bcl-2 activity, can interact with VDAC to increase VDAC aperture and increases mitochondrial permeability, promoting apoptosis [71]. The proper proportion of Bax and Bcl-2 maintains the cell homeostasis to ensure cell survival [72].

Lemasters et al. [67] introduced a concept of aldehyde-dependent VDAC closure, mitochondrial uncoupling, and disruption of normal mitochondria functioning resulting from ethanol metabolism effects. Aldehydes derivation during lipid peroxidation, VDAC closure is probably a common feature leading to liver pathologies as was pointed out on almost indistinguishable histopathological manifestations in alcoholic liver disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and toxicant-associated steatohepatitis. Ethanol is metabolized predominantly by the liver in two-step oxidation: first to acetaldehyde (AcAld) by catalytic action of alcohol dehydrogenase (ALD) followed by oxidation to acetate by aldehyde dehydrogenase (ALDH). The first step undergoes in cytosol and peroxisomes by effect of cytochrome P4502E1 and catalase. Although the oxidation is prevalent over ALD, the consequence of cytochrome P450 metabolism is overwhelmingly negative due to the formation of ROS, e.g., hydroxyethyl radicals. Of the 19 known mammalian ALDH genes, mitochondrial ALDH2 with high affinity for AcAld (Km < 1 μM) is the most important for AcAld oxidation (and detoxification) to acetate [73]. In both reactions, by oxidation of 1 mole of ethanol, 2 moles of NADH are formed, further requiring oxidation in the respiratory chain. As little as 2.5 h after a single bolus dose of ethanol ingestion a swift increase of alcohol metabolism (SIAM), an adaptive increase of hepatic ethanol metabolism occurs [74]. Mitochondrial respiration causing NADH oxidation nearly doubles, but it does not lead to increased ATP generation. To the contrary, hepatic ATP decreases, glycolysis is stimulated, and glycogen stores are depleted. Furthermore, mitochondrial β-oxidation of fatty acids becomes inhibited, promoting fat accumulation within hepatocytes (steatosis) [75]. This is probably the result of decrease in MOM permeability most likely by VDAC closure, promoting selective oxidation of AcAld, since VDAC closure blocks mitochondrial ATP release, respiratory substrates uptake, and uptake of fatty acids for β-oxidation [67]. Adrenergic hormones release free fatty acids from adipose tissue, which serve as substrates for long-chain fatty acid peroxisomal β-oxidation. The ensuing peroxisomal H<sup>2</sup> O2 formation then can promote catalase-dependent alcohol metabolism [67]. AcAld is toxic to mitochondria and aggravates oxidative stress by binding to GSH and promoting GSH leakage [76]. Moreover, as mentioned before, ethanol metabolism and also NADH overproduction cause formation of ROS, lipid peroxidation, onset of the mitochondrial permeability transition, and apoptosis [77]. However, as has been showed, short- and medium-chain fatty acids can cross mitochondrial membrane freely using carnitine shuttle or other transport system [78] and therefore are presented in diet capable to prevent steatosis development.

Tom7 commonly occur in eukaryotic organisms [82]. To prevent misfolding and aggregation, hydrophobic segments of mitochondrial precursor proteins are shielded in cytosol by chaperones that escort them to the mitochondria surface [83]. These chaperones are Hsp70 (and its partner J proteins), Hsp90, and mitochondrial import stimulation factor (MSF) [84]. MSF with precursor proteins loaded was suggested to bind to Tom70 and forward proteins to Tom20, with ATP consumption, while Hsp70 transfers proteins to Tom70 without ATP hydrolysis [85]. In addition, it was found that mitochondrial proteins are also allowed to bind Tom20 directly without Hsp70 [86]. Except for some α-helical outer membrane proteins, virtually all precursors initially enter mitochondria by passing a TOM complex. As also discussed by Dudek et al. [2], many IMS proteins are imported by the mitochondrial intermembrane space import and assembly (MIA) machinery, which couples sorting of client proteins to their oxidative folding through a disulfide bridge formation. Small Tim chaperones of IMS bound to outer membrane β-barrel proteins transfer preproteins to the sorting and assembly machinery (SAM), allowing their integration into the MOM. Tim chaperones also convoy mitochondrial metabolite carrier proteins through the IMS, which are then integrated into the MIM by translocase of the inner membrane 22 (TIM22) complex in a membrane potential–driven manner. Preproteins of matrix-targeted proteins are directly transferred from TOM to TIM23 (presequence translocase) without IMS chaperones, ensuring their translocation into the matrix or later sorting into the MIM. The only source of energy for lateral membrane integration is membrane potential. Complete import of preproteins into the matrix requires the ATP-

Nuclear Encoded Mitochondrial Proteins in Metabolite Transport and Oxidation Pathway…

http://dx.doi.org/10.5772/intechopen.72937

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dependent presequence translocase-associated import motor (PAM).

(deficiency in Hsp60 causing decrease in mitochondrial metabolic pathways).

The inner mitochondrial membrane is relatively low permeable to ions in order to minimize energy dissipation formed on complexes through generation of electrochemical proton gradient, in its direct link with ADP phosphorylation. Random flow of charged metabolites

**6. Mitochondrial inner membrane carriers**

Affection by the TOM40 complex functions leads to dysfunction of mitochondria and oxidative damage and is in the background of misfolding protein diseases. Bender et al. [87] found a significantly reduced TOM40 in the brain of Parkinson's disease patients in connection with increased mtDNA damage and α-synuclein transgenic mice together with altered levels of complex I proteins. The stable complexes of accumulated truncated amyloid precursor protein and TOM40 cause mitochondrial dysfunction in brains of Alzheimer disease patients [88]. Other diseases in humans associated with abnormal mitochondrial transport of proteins, as reviewed in MacKenzie and Payne [89], are primary hyperoxaluria type I (caused by alanine/ glyoxylate aminotransferase 1 deficiency), pyruvate dehydrogenase deficiency, susceptibility to severe alcoholic liver disease (caused by increased Ala-MnSOD activity due to inability to transfer Val-MnSOD through MIM), deafness dystonia syndrome (caused by mutations in IMS protein deafness dystonia peptide 1), dilated cardiomyopathy with ataxia (caused by dysfunctional import of matrix proteins through the TIM23 complex), spastic paraplegia (dysfunction of Hsp60), and atypical mitochondria disease involving multisystem failure

#### **5.1. Translocase of the MOM**

The endosymbiotic relationship of α-proteobacteria and archaic eukaryotic cell results in massive loss and transfer of coding sequences from mtDNA to the nucleus and only less than 1% is retained in today's mtDNA. Thus, most mitochondrial proteins (1000–1500) undergo cytosol translation and are subsequently transferred to mitochondria, requiring membrane complexes of protein translocators, translocases, or translocons. They include TOM and TIM for large conductance channels with almost identical properties [79]. In addition, other mitochondria protein translocators like TOB/SAM complex in MOM and Mia40/Tim401-Erv1 redox translocator in MIM have been identified [80, 81].

The general entry gate for mitochondrial proteins is thought to be TOM40 complex in MOM consisting of core sequence Tom40, Tom22, Tom7, Tom6, Tom5, peripheral associated receptors Tom20, Tom70, and a minor component Tom71. Among them, only Tom40, Tom22, and Tom7 commonly occur in eukaryotic organisms [82]. To prevent misfolding and aggregation, hydrophobic segments of mitochondrial precursor proteins are shielded in cytosol by chaperones that escort them to the mitochondria surface [83]. These chaperones are Hsp70 (and its partner J proteins), Hsp90, and mitochondrial import stimulation factor (MSF) [84]. MSF with precursor proteins loaded was suggested to bind to Tom70 and forward proteins to Tom20, with ATP consumption, while Hsp70 transfers proteins to Tom70 without ATP hydrolysis [85]. In addition, it was found that mitochondrial proteins are also allowed to bind Tom20 directly without Hsp70 [86]. Except for some α-helical outer membrane proteins, virtually all precursors initially enter mitochondria by passing a TOM complex. As also discussed by Dudek et al. [2], many IMS proteins are imported by the mitochondrial intermembrane space import and assembly (MIA) machinery, which couples sorting of client proteins to their oxidative folding through a disulfide bridge formation. Small Tim chaperones of IMS bound to outer membrane β-barrel proteins transfer preproteins to the sorting and assembly machinery (SAM), allowing their integration into the MOM. Tim chaperones also convoy mitochondrial metabolite carrier proteins through the IMS, which are then integrated into the MIM by translocase of the inner membrane 22 (TIM22) complex in a membrane potential–driven manner. Preproteins of matrix-targeted proteins are directly transferred from TOM to TIM23 (presequence translocase) without IMS chaperones, ensuring their translocation into the matrix or later sorting into the MIM. The only source of energy for lateral membrane integration is membrane potential. Complete import of preproteins into the matrix requires the ATPdependent presequence translocase-associated import motor (PAM).

Affection by the TOM40 complex functions leads to dysfunction of mitochondria and oxidative damage and is in the background of misfolding protein diseases. Bender et al. [87] found a significantly reduced TOM40 in the brain of Parkinson's disease patients in connection with increased mtDNA damage and α-synuclein transgenic mice together with altered levels of complex I proteins. The stable complexes of accumulated truncated amyloid precursor protein and TOM40 cause mitochondrial dysfunction in brains of Alzheimer disease patients [88]. Other diseases in humans associated with abnormal mitochondrial transport of proteins, as reviewed in MacKenzie and Payne [89], are primary hyperoxaluria type I (caused by alanine/ glyoxylate aminotransferase 1 deficiency), pyruvate dehydrogenase deficiency, susceptibility to severe alcoholic liver disease (caused by increased Ala-MnSOD activity due to inability to transfer Val-MnSOD through MIM), deafness dystonia syndrome (caused by mutations in IMS protein deafness dystonia peptide 1), dilated cardiomyopathy with ataxia (caused by dysfunctional import of matrix proteins through the TIM23 complex), spastic paraplegia (dysfunction of Hsp60), and atypical mitochondria disease involving multisystem failure (deficiency in Hsp60 causing decrease in mitochondrial metabolic pathways).
