**3.3. Post-translational modifications of proteins during aging**

software and using *gene IDs* (**Figure 5**). Two proteins, methylmalonate-semialdehyde dehydrogenase [acylating] (Gene ID: *Aldh6a1*) and carnitine O-palmitoyltransferase 2 (*Cpt2*) did not fit to the protein-protein interaction map. However, their role in fatty acid metabolism is very important in connection to the heart muscle work and energy production. The rest of the proteins participate in various metabolic pathways of mitochondria. Three take part in Krebs cycle – malate dehydrogenase (*Mdh1*), dihydrolipoyl dehydrogenase (*Dld*) and aconitate hydratase (*Aco2*). Post-translational cysteine-related modification of aconitase was reported to be a key in linkage between Krebs cycle, redox signaling and metabolism of ROS [60]. Next five down-regulated proteins were directly connected with cardiac muscle contraction and the risk of developing age-related neuronal disorders - Huntington, Parkinson and Alzheimer disease. All of them are subunits of ETC, thus regulate production of energy in mitochondria: NADH dehydrogenase 1 alpha subcomplex subunit 5 (*Ndufa5*), cytochrome bc1 complex subunit 1 (*Uqcrc1*) and cytochrome bc1 complex subunit Rieske (*Uqcrsf2*), cytochrome c oxidase subunit 5B (*Cox5b*), electron transfer flavoprotein subunit beta (*Etfb*). Combined defects in oxidative phosphorylation and fatty acid beta oxidation were detected in mitochondrial diseases [61]. Creatine kinase M-type (*Ckm*) was only one cytoplasmic enzyme interacting with mitochondria through transfer of phosphate and the last one is voltage-dependent anion-selective channel protein 1 (*Vdac1*),

72 Mitochondrial Diseases

**Figure 5.** Functional protein-protein interactions during aging in rat mitochondria (yet unpublished data).

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Global analyses of enzymes using proteomics revealed widespread distribution of PTMs on many key enzymes located in all cellular compartments. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Enzymatic PTMs can be detected in ever increasing amounts and they appear to be critical for folding and assembly (e.g. glycosylation), function as key regulators of catalytic activity of enzymes (e.g. binding of prosthetic groups, phosphorylation), or mark enzyme molecules for targeted destruction (e.g. ubiquitylation). In parallel with these processes there are non-enzymatic PTMs caused by ROS and RNS continuously interacting with individual enzyme molecules. These PTMs contribute to molecular aging and may also be involved in regulation of enzymes' catalytic activity [64]. There are two groups of PTMs observed during oxidative stress mediated aging, reversible and irreversible. The major types of *irreversible PTMs* are carbonylation and 3-nitrotyrosilation.


Second group of *reversible PTMs* which are related to the aging process are sulfur-mediated S-Sulfenylation, S-nitrosylation, S-glutathionylation and lipid peroxidation pathway-related HNE modification.

• Protein *S-Sulfenylation* leads to the production of sulfur-hydroxylation product (P-SOH), disulfide bond and sulfenyl-amide bond formation. It may be a precursor to the process of S-sulfinylation and S-sulfonylation. This type of modification is believed to be a fleeting molecular switch that regulates non-enzymatic oxidative folding [68].

suggesting that under basal resting conditions, one mitochondrion per cell is replaced every 40 minutes [73]. In order to facilitate and initiate mitophagy, mitochondrial fusion and fission play a critical role in mitochondrial turnover. Fission of mitochondria into smaller fragments is a crucial requirement for mitophagy to occur. The key regulator of this process is dynamin-related protein 1 (Drp1), which in concert with proteins fission 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 kDa (MiD49) and 51 kDa (MiD51) is responsible for mitochondrial fragmentation. The role of last three proteins appears essential. Mff assists in the assembly of Drp1. MiD49 and MiD51 may play a regulatory role by recruiting Drp1 and maintaining it in inactive state until fission is required [74]. Proteins that promote outer mitochondrial membrane (OMM) fusion such as Mitofusin 1 and 2 (Mfn1 and 2) are ubiquitinated and eliminated by the ubiquitin proteasome system. E3 ubiquitin ligase Parkin (also known as Park2) and PTEN-induced putative kinase 1 (PINK1) have been shown to play an important role in mitophagy. PINK1 targets to the mitochondria but is normally degraded by presenilin associated rhomboid-like protease (PARL). In response to loss of mitochondrial membrane potential, PARL is inactivated; PINK1 is stabilized and recruits Parkin. Parkin ubiquitinates several mitochondrial associated proteins and they are then recognized by p62 and bring mitochondria to the autophagosomes. Thus mitochondria with membrane potential loss can be selectively degraded. Parkin substrates include e.g. voltage-dependent anion-selective channel protein (VDAC), translocase of the outer membrane (TOM), mitochondrial fission 1 (FIS1), hexokinase, mitochondrial Rho-GTPase (MIRO) 1 and 2, although whether ubiquitination of these proteins is required or sufficient for mitophagy is unclear and highly dependent on the specific cellular

Interplay Between Mitochondrial Proteins and Age-Associated Risk of Cardiovascular Diseases

http://dx.doi.org/10.5772/intechopen.71789

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The importance of mitophagy for the preservation of cardiovascular homeostasis, the cardiomyocyte-specific deletion of Parkin and the expression of a mutant Mfn2 (mitofusin 2) at birth prevented the switch from fetal to adult mitochondria in the mice heart [76]. Another example, mice bearing a heart-specific deletion of Mfn2 prematurely succumbed to a progressive cardiomyopathy characterized by impaired contractile function [77]. Such a detrimental phenotype could be reversed, at least partially by Mfn2 to prevent the targeted mitochondria from rejoining the mitochondrial network through fusion. Under basal conditions Mfn2 functions in mitochondrial fusion events and links endoplasmic reticulum to mitochondria. Also acts as Parkin receptor during mitophagy following phosphorylation by PINK1 and recruiting Parkin to the mitochondria. So even in case of Parkin-dependent mitophagy, some outer mitochondrial membrane proteins are recycled through transfer to the endoplasmic reticulum. Most studies have relied on the systemic modulation of autophagy with nutritional or pharmacological interventions or the homozygous/heterozygous deletion of a relevant gene. Nutritional and pharmacological interventions commonly used to modulate autophagy in the cardiovascular system *in vivo*, including caloric restriction or caloric restriction mimetics, rapamycin, 3-methyladenine, and lysosomal inhibitors, are rather nonspecific. Genetic interventions offer increased specificity, but are not devoid of potential problems that should be kept under attentive consideration. Linking mitophagy with cardiomyocytes is the field of interest in many publications [78]. While mitophagy is responsible for bulk degradation of mitochondria, turnover of individual components may proceed at asynchronous rates

context [75].


Above mentioned PTMs occur the most frequently during oxidative stress-related aging process. However, there are virtually hundreds of additional PTMs that may occur in enzymes. To support results from protein profiling of mitochondria is important to focus on deep protein analyzes of individual selected proteins, their interactions within the individual compartments, between different organelles or the intercellular communication. For this purpose, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies.
