**3.2. Mitochondrial protein-protein network**

old rat hearts (**Figure 3**). Low Trx level is probably result of TrxR malfunctioning due to lack of NADPH, overproduction of ROS and/or RNS. Thioredoxin also plays a role in the reversible S-nitrosylation of cysteine residues in target proteins, and thereby contributes to the response of intracellular nitric oxide (NO). In addition, Trx is able to block caspase-3 activity through nitrosylation of the active cysteine site in response to NO. Therefore, Trx protein deficiency may contribute to the stimulation of caspase-dependent apoptosis. The most studied enzyme in the process of protein S-glutathionylation is glutaredoxin (Grx). The high specificity of Grx to S-glutathionylated proteins is used as a tool for studying and identifying them. In general, it is stated that the main task of Grx is to remove GSH from S-glutathionylated proteins. Thus, reduced thiol-containing protein is restored, which was confirmed in experiments with siRNA (small interfering RNA). The suppression of Grx genes with siRNA is an approach to study not only the "antioxidant" properties of Grx but also the role of protein S-glutathionylation [58]. Grx2 catalyzes S-glutathionylation of IMM proteins in a relatively reduced GSH/GSSG ratio equal to 6. The fact that thiol-disulfide oxidoreductases can catalyze both oxidizing and reducing reactions is not exclusive to Grx. Thioredoxin can act as an oxidant in oxidizing environment. Currently, the biggest challenge of researchers is to identify which enzymes are responsible for (de)glutathionylation, and if it is spontaneous process or catalyzed by enzymes. Existing information suggests that the

**Figure 3.** Scheme of (A) Thiol-disulfide network and (B) protein level for Prx2, Trx (yet unpublished data). GSH-reduced glutathione, GSSG-oxidized glutathione, GPx-Glutathione peroxidase, GR-Glutathione reductase, Grx-Glutaredoxin, Prx-Peroxiredoxin, Trx-Thioredoxin, TrxR-Thioredoxin reductase, SH-reduced and SS-oxidized form of Protein, ICDH-

isocitrate dehydrogenase.

70 Mitochondrial Diseases

One of the major goals of gerontology is to understand the comprehensive mechanisms involved in aging at different levels and hopefully to help understand age-related diseases. The complexity of the proteome supersedes that of the genome, due to alternative splicing events and PTMs of proteins. Proteomes are expected to be two to three orders of magnitude more complex than would be predicted from numbers of protein-encoding genes present in the respective genomes [59]. It is widely recognized that cellular aging causes changes in the proteome. However, the nature and targets of these changes and their consequences have not yet been completely identified. In recent years, mass spectrometry (MS) has been recognized as a golden standard tool for the identification and analysis of individual proteins. For further understanding of the molecular changes during heart aging, we have identified several proteins and compared the differences in the mitochondrial protein expression profiles among two age groups.

Precipitated proteins from mitochondria of 6 and 27 months old rat hearts were separated with two-dimensional electrophoresis (2-DE) to provide a protein profile (**Figure 4**). Interestingly, the change in protein level was statistically significant (1.5-fold change, 95% confidence interval) in only 12 proteins (marked with red circles) from the total protein pool of mitochondria. All the proteins were down-regulated in senescent mitochondria in comparison to the adult ones. Despite the small number of quantitatively modified proteins, these create an interesting protein-protein network. The strength of data support represents line thickness of protein-protein interaction network generated by String

**Figure 4.** Representative 2-DE analysis of mitochondrial proteins in aging rat heart (yet unpublished data).

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*),

which plays a role in outer mitochondrial membrane permeabilization and cellular death. This outer mitochondrial membrane protein is tightly bound with Alzheimer disease, where mediates amyloid β toxicity and represents a potential target for Alzheimer disease

Currently available evidence indicates that the steady-state amounts of structural damage to proteins accumulated during life are relatively small, and are often present only in trace amounts. On the other hand, the pro-oxidizing changes in the redox state reflected by the

tions are significant and ubiquitous. However, the oxidative PTMs are relevant only if these are connected to functional consequences. It is important to consider that a slight modification in low abundance proteins may be of physiological importance. Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical/ biophysical analysis of target proteins [63]. Thus, proteomic approaches represent powerful tools to address these questions by identifying the targeted proteins and the extent of their

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

• *Carbonylation* is covalent adduction of lipid aldehyde to the side chains of lysine, histidine or cysteine residues. Extensive amount of information about this modification can be found

• *3-nitrotyrosilation*, frequently called tyrosine nitration is formed between RNS (peroxynitrite anion) and a tyrosine residue of target protein. Extensive research was done during

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

O2

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

and level of protein modifica-

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

73

decline in redox potential, increases in production of H2

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

therapy [62].

modifications.

3-nitrotyrosilation.

HNE modification.

in the recently published book [65].

last years in failing human [66] and rat hearts [67].

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

which plays a role in outer mitochondrial membrane permeabilization and cellular death. This outer mitochondrial membrane protein is tightly bound with Alzheimer disease, where mediates amyloid β toxicity and represents a potential target for Alzheimer disease therapy [62].

Currently available evidence indicates that the steady-state amounts of structural damage to proteins accumulated during life are relatively small, and are often present only in trace amounts. On the other hand, the pro-oxidizing changes in the redox state reflected by the decline in redox potential, increases in production of H2 O2 and level of protein modifications are significant and ubiquitous. However, the oxidative PTMs are relevant only if these are connected to functional consequences. It is important to consider that a slight modification in low abundance proteins may be of physiological importance. Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical/ biophysical analysis of target proteins [63]. Thus, proteomic approaches represent powerful tools to address these questions by identifying the targeted proteins and the extent of their modifications.
