**3. The age-dependent attenuation of bioenergetics underlies a decrease in the general level of protein synthesis**

#### **3.1 Introduction**

A different popular aging theory, the protein-error theory, is based on the indisputable fact that the bulk protein synthesis slows down during aging (Rattan, 1996, 2009; Ryazanov & Nefsky, 2002). According to the theory, such retardation results in a decreasing protein turnover rate which causes the accumulation of defective macromolecules. S.I.S. Ratton (1996), who has investigated this process in detail, reports that "the implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters and the components of the extra cellular matrix." The reason behind a slower protein synthesis rate is seen in the stochastic accumulation of molecular damage and the progressive failure of maintenance and repair (Rattan, 2009). It entails damage to fragments of the translation mechanism: "a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors" (Rattan, 2006). At the same time, there is empirical evidence which allows for the explanation of the slowing down of overall protein synthesis by the attenuation of cellular bioenergetics.

#### **3.2 The mechanism for the decrease in the level of cell protein synthesis**

It was D.A. Young (1969) who discovered a relationship between the protein synthesis rate and the bioenergetics level for the first time. When conducting experiments on glucocorticoid hormones, he noticed that the rate of amino acids inclusion into a growing polypeptide chain depends upon the entry of carbohydrates (glucose, pyruvate, and lactate) into cells. An assumption was made that this effect is connected with the ATP generation rate. It was shown thereafter that the protein synthesis rate depends upon the ADP/ATP and GDP/GTP ratios

Bioenergetics Theory of Aging 71

The molecular mechanism of protein synthesis is currently well-understood and has been detailed in a number of reviews (Pain, 1996; Rattan, 2009). It was shown that the protein synthesis rate for eukaryotes is controlled at the translation level (Hucul, et al., 1985; Kimball et al., 1998). Among three translation stages (initiation, elongation and termination), the regulatory stage is the initiation (Hucul, et al., 1985; Kimball et al., 1998). The goal of this stage is the sequential binding of first the 40s and then the 60s ribosomal subunit to a messenger RNA molecule. At least 12 recirculation eukaryotic initiation factors (eIF) are involved in this stage. The initiation process can be divided into three phases (Fig. 2): (1) the association of the Met-tRNA initiator and several initiation factors with the 40s ribosomal subunit so as to form the pre-initiation complex; (2) the binding of this complex to a messenger RNA (mRNA) molecule, and (3) the addition of the 60s ribosomal subunit to

The first initiation phase starts with the binding of the Met-tRNA initiator to a pre-existing double complex eIF2GTP. When this preinitiation complex binds to mRNA at the second phase, GTP is oxidised to form GDP, and the oxidation energy is used to create bonds, with inorganic phosphorus being released. At the third stage, when the goal has been reached, the preinitiation complex disintegrates into separate initiation factors; these factors are then recycled to catalyse further initiation events. eIF2 is released as a binary complex with GDP, which is stable but not functionally active, i.e. it is unable to bind to a new Met-tRNA. A guanine nucleotide exchange factor, eIF2B, is required to catalyse the regeneration of the eIF2GTP. Energy for such regeneration is provided by ATP oxidation to form ADP and the ADP is then reduced in the bioenergetics machine. Thus, the total protein synthesis level is originally regulated by the eIF2 recirculation rate which, in turn, depends upon the cellular

If the GDP-to-eIF2GDP reduction is interrupted, the protein synthesis in the cell is blocked (Clemens, 1994). The natural mechanism protecting an organism in various stressful situations is based on this phenomenon: the phosphorylating of α-subunit eIF2 by different specific protein kinases blocks the reaction of the GDP-to-GTP exchange, which results in a complete protein synthesis termination in the cell followed by apoptosis (Clemens, 1994; Clemens et al., 2000). Such specific protein kinases are expressed in the cell when emergencies occur, such as an occurrence of the double-stranded replicative form of viral RNA (Jeffrey et al. 2002; Pain, 1996; Robert et al., 2006), irreparable damage of the genetic apparatus (Zykova et al., 2007; Jeffrey et al, 2002), acute shortage in amino acids (Clemens et al., 2001; Harmon et al., 1984), and malignant cell transformation (Clemens, 1994, Mendelsohn et al., 1977). Under normal physiological conditions when there are no specific protein kinases, the GDP-to-GTP exchange rate in the eIF2GDP complex (and, consequently, the total protein synthesis rate) is regulated

Programmed bioenergetics decline is the original cause of overall protein synthesis decrease

Tissue senility is the most visible phenomenon and one of the most harmful phenomena of organism aging. Its cause was determined half a century ago (Hayflick & Moorhead, 1961):

assemble an 80s ribosome at the initiation codon.

by the cellular bioenergetics (Hucull et al., 1985).

rather than the stochastic accumulation of molecular damage.

**4. The Hayflick limit is caused by the age-related decrease in the** 

bioenergetics value.

**3.3 Conclusion** 

**bioenergetics level** 

**4.1 Introduction** 

rather than on the absolute ATP value (Hucull et al., 1985; Mendelsohn et al., 1977; Young, 1970). In these tests, minor changes in the nucleotide diphosphate /nucleotide triphosphate ratio resulted in a significant effect on the range corresponding to a physiological energy level. The authors came to the conclusion that the ADP/ATP and/or GDP/GTP ratios are a physiological regulator of the protein synthesis rate.

The initiation of translation can be divided into three phases. Phase one: the initiator methionine transport RNA (Met-tRNA) binds with the pre-existing binary complex eIF2·GTP and the 40S ribosomal subunit to provide the pre-initiation complex 40S·Met-tRNA·eIF2·GTP. Phase two: the pre-initiation complex binds to messenger RNA (mRNA). When the pre-initiation complex stops at the initiation codon of the mRNA, the GTP molecule is hydrolysed to GDP, inorganic phosphorus (Pi) is liberated and the energy of oxidation is spent on bond formation. This powers the ejection of the factors bound to the 40S ribosomal subunit in the third phase. The continuity of the initiation of these events requires the recycling of initiation factor molecules. eIF2 is released as an inactive binary complex with GDP and requires a guanine nucleotide exchange factor, eIF2·B, to catalyse regeneration of the eIF2GTP. Energy support of regeneration is carried out at the expense of GTP oxidation. The GDP formed is then reduced at the expense of ATP oxidation in a reaction catalysed by nucleoside diphosphate kinase (NDK). The ADP formed is in turn reduced to ATP in the mitochondrial bioenergetics machine. The programmed bioenergetics decline decreases the eIF2 recirculation rate and thus reduces the general level of protein synthesis.

Fig. 2. The simplified scheme for the initiation of translation and its connection with bioenergetics.

rather than on the absolute ATP value (Hucull et al., 1985; Mendelsohn et al., 1977; Young, 1970). In these tests, minor changes in the nucleotide diphosphate /nucleotide triphosphate ratio resulted in a significant effect on the range corresponding to a physiological energy level. The authors came to the conclusion that the ADP/ATP and/or GDP/GTP ratios are a

The initiation of translation can be divided into three phases. Phase one: the initiator methionine transport RNA (Met-tRNA) binds with the pre-existing binary complex eIF2·GTP and the 40S ribosomal subunit to provide the pre-initiation complex 40S·Met-tRNA·eIF2·GTP. Phase two: the pre-initiation complex binds to messenger RNA (mRNA). When the pre-initiation complex stops at the initiation codon of the mRNA, the GTP molecule is hydrolysed to GDP, inorganic phosphorus (Pi) is liberated and the energy of oxidation is spent on bond formation. This powers the ejection of the factors bound to the 40S ribosomal subunit in the third phase. The continuity of the initiation of these events requires the recycling of initiation factor molecules. eIF2 is released as an inactive binary complex with GDP and requires a guanine nucleotide exchange factor, eIF2·B, to catalyse regeneration of the eIF2GTP. Energy support of regeneration is carried out at the expense of GTP oxidation. The GDP formed is then reduced at the expense of ATP oxidation in a reaction catalysed by nucleoside diphosphate kinase (NDK). The ADP formed is in turn reduced to ATP in the mitochondrial bioenergetics machine. The programmed bioenergetics decline decreases the eIF2

Fig. 2. The simplified scheme for the initiation of translation and its connection with

recirculation rate and thus reduces the general level of protein synthesis.

bioenergetics.

physiological regulator of the protein synthesis rate.

The molecular mechanism of protein synthesis is currently well-understood and has been detailed in a number of reviews (Pain, 1996; Rattan, 2009). It was shown that the protein synthesis rate for eukaryotes is controlled at the translation level (Hucul, et al., 1985; Kimball et al., 1998). Among three translation stages (initiation, elongation and termination), the regulatory stage is the initiation (Hucul, et al., 1985; Kimball et al., 1998). The goal of this stage is the sequential binding of first the 40s and then the 60s ribosomal subunit to a messenger RNA molecule. At least 12 recirculation eukaryotic initiation factors (eIF) are involved in this stage. The initiation process can be divided into three phases (Fig. 2): (1) the association of the Met-tRNA initiator and several initiation factors with the 40s ribosomal subunit so as to form the pre-initiation complex; (2) the binding of this complex to a messenger RNA (mRNA) molecule, and (3) the addition of the 60s ribosomal subunit to assemble an 80s ribosome at the initiation codon.

The first initiation phase starts with the binding of the Met-tRNA initiator to a pre-existing double complex eIF2GTP. When this preinitiation complex binds to mRNA at the second phase, GTP is oxidised to form GDP, and the oxidation energy is used to create bonds, with inorganic phosphorus being released. At the third stage, when the goal has been reached, the preinitiation complex disintegrates into separate initiation factors; these factors are then recycled to catalyse further initiation events. eIF2 is released as a binary complex with GDP, which is stable but not functionally active, i.e. it is unable to bind to a new Met-tRNA. A guanine nucleotide exchange factor, eIF2B, is required to catalyse the regeneration of the eIF2GTP. Energy for such regeneration is provided by ATP oxidation to form ADP and the ADP is then reduced in the bioenergetics machine. Thus, the total protein synthesis level is originally regulated by the eIF2 recirculation rate which, in turn, depends upon the cellular bioenergetics value.

If the GDP-to-eIF2GDP reduction is interrupted, the protein synthesis in the cell is blocked (Clemens, 1994). The natural mechanism protecting an organism in various stressful situations is based on this phenomenon: the phosphorylating of α-subunit eIF2 by different specific protein kinases blocks the reaction of the GDP-to-GTP exchange, which results in a complete protein synthesis termination in the cell followed by apoptosis (Clemens, 1994; Clemens et al., 2000). Such specific protein kinases are expressed in the cell when emergencies occur, such as an occurrence of the double-stranded replicative form of viral RNA (Jeffrey et al. 2002; Pain, 1996; Robert et al., 2006), irreparable damage of the genetic apparatus (Zykova et al., 2007; Jeffrey et al, 2002), acute shortage in amino acids (Clemens et al., 2001; Harmon et al., 1984), and malignant cell transformation (Clemens, 1994, Mendelsohn et al., 1977). Under normal physiological conditions when there are no specific protein kinases, the GDP-to-GTP exchange rate in the eIF2GDP complex (and, consequently, the total protein synthesis rate) is regulated by the cellular bioenergetics (Hucull et al., 1985).

#### **3.3 Conclusion**

Programmed bioenergetics decline is the original cause of overall protein synthesis decrease rather than the stochastic accumulation of molecular damage.

## **4. The Hayflick limit is caused by the age-related decrease in the bioenergetics level**

#### **4.1 Introduction**

Tissue senility is the most visible phenomenon and one of the most harmful phenomena of organism aging. Its cause was determined half a century ago (Hayflick & Moorhead, 1961):

Bioenergetics Theory of Aging 73

erosion is accepted. Therefore, the question of the Hayflick limit's nature is without answer. Apparently, an alternative reason for this phenomenon should be looked for in the

The cell division cycle (proliferative cycle) is divided into four phases (Sherr, 1994): G1, S,

Cycle of cell reduplication is divided into 4 phases: G1 (gap 1), S (synthesis), G2 (gap 2), and M (mitosis).

In the G1-phase, precursor molecules necessary for DNA reduplication and doubling of all cell structures in the following division are synthesized. In the following S-phase, DNA is reduplicated, and after a short G2-phase, a cell enters M. Numerous studies have showed that all non dividing cells stay in the G1-phase. If a cell has passed through this phase, then it will pass through other phases automatically with almost equal speed. As far as entry of old cells into irreversible proliferative quiescence is concerned, we will be interested only in events occurring in the G1-phase. Control of the cell division rate is performed by endogenous and exogenous (for a cell) regulatory factors that are stimulators and inhibitors of proliferation. As an example of such regulation, the data of one of the first works in this field (Leof, et al., 1982) accurately reflecting the essence of the phenomenon are shown. The effect of different growth factors on mouse fibroblasts was studied. It was shown that, right after mitosis completion, a cell enters a proliferative quiescence state between the M and G1 phases (the G0-phase). To be removed from this state, a cell needed an external proliferative signal from platelet derived growth factor (PDGF). No structural or biochemical changes in a quiescent cell happened without the signal, and it remained insensitive to other proliferative stimuli. This primary stimulus is a competence factor. After a cell has received

**4.3 The reason for termination of proliferation of old cells** 

mechanism of cell division.

Fig. 3. Phases of proliferative cell cycle.

G2, and M (Fig.1).

higher eukaryotic cells do not divide infinitely, and, after a certain number of doublings, they enter a nondividing but viable state. Human fibroblasts, for example, are able to divide 53 ± 6 times over 302 ± 27 days and be in a stationary state for 305 ± 41 more days (Bayreuther et al., 1988). This limitation of division, the Hayflick limit, underlies the replicative aging theory, which is recognized to be one of the most striking modern aging theories (Anisimov, 2003). The main postulate of this theory is that, due to accumulation of old nondividing cells, tissue renewing homeostasis is violated, which causes their degradation (Hornsby, 2002; Itahana et al., 2004; Yegorov & Zelenin, 2003).

#### **4.2 Modern views on the cause of cell proliferation limitation**

A convincing mechanism of termination of old cells division was predicted theoretically by A.M. Olovnikov in 1971 and then confirmed experimentally (Greider & Blackburn, 1985). Vertebrates' chromosome ends from the DNA 3'-end have repeating nucleotide sequences telomeres. They prevent fusion of chromosome ends, protect DNA from nuclease digestion, and participate in doubled chromosome disjunction in mitosis. In embryonic cells telomeres are synthesized by a special enzyme telomerase, which most somatic cells do not have. Because of the necessity of RNA-primer during DNA reduplication initiation, the telomere ends of somatic cells chromosomes are shortened with every cycle. As a result, after a certain number of doublings, the telomere end is depleted and divisions are terminated due to chromosome erosion (Itahana et al., 2004). This mechanism was confirmed by numerous empirical facts: 90–95% of potentially immortal cancer cells possess telomerase activity and the telomere end of their chromosomes is not shortened; suppression of telomerase activity in these cells causes shortening of the telomere end and division termination, i.e., aging; and restoration of telomerase activity makes them potentially immortal again. Therewith, facts contradictory to this conception were accumulated. The most convincing of them were obtained by a research group led by Blasco (Blasco et al., 1997). They obtained mice zygotes lacking a telomerase gene but with full-sized initial chromosome telomere ends. Mice developed from these zygotes were not only viable, but also fertile. This initial telomere length was sufficient to maintain normal viability of six mouse generations. In the first generation, for example, mice passed through youth and maturity successfully and died in old age having 80% of telomeres in reserve. Only in the fifth and sixth generations did anomalies caused by chromosome telomere end depletion appear. These data were confirmed by another group of authors led by Herrera (Herrera et al., 1999). They obtained an analogous mouse line, but with a shortened initial telomere end, and repeated the experiments of Blasco et al. These mice were viable for only four generations, and anomalies in late generations were related with depletion of telomeres in cells of tissues with the most intensive proliferation (Lee et al., 1998). By the present time, researchers of the telomere mechanism incline to the conclusion that loss of the telomere end indeed leads to chromosome erosion and cell death, but cell proliferation termination during normal physiological cell aging happens earlier than this critical moment and a cell that has expended all its proliferative potential still contains a significant telomere reserve. The telomere mechanism serves as an additional barrier on the road to reproduction of malignant cells (Itahana et al., 2004). The conclusion that there is nonparticipation of the telomere apparatus in the mechanism of termination of old cells' division could have been drawn from the very beginning. It followed from the results of the initial Hayflick experiments that, after a certain number of doublings, a cell enters a nondividing, but viable, state, and there is no sense in discussing viability if division termination due to chromosome

higher eukaryotic cells do not divide infinitely, and, after a certain number of doublings, they enter a nondividing but viable state. Human fibroblasts, for example, are able to divide 53 ± 6 times over 302 ± 27 days and be in a stationary state for 305 ± 41 more days (Bayreuther et al., 1988). This limitation of division, the Hayflick limit, underlies the replicative aging theory, which is recognized to be one of the most striking modern aging theories (Anisimov, 2003). The main postulate of this theory is that, due to accumulation of old nondividing cells, tissue renewing homeostasis is violated, which causes their degradation (Hornsby, 2002;

A convincing mechanism of termination of old cells division was predicted theoretically by A.M. Olovnikov in 1971 and then confirmed experimentally (Greider & Blackburn, 1985). Vertebrates' chromosome ends from the DNA 3'-end have repeating nucleotide sequences telomeres. They prevent fusion of chromosome ends, protect DNA from nuclease digestion, and participate in doubled chromosome disjunction in mitosis. In embryonic cells telomeres are synthesized by a special enzyme telomerase, which most somatic cells do not have. Because of the necessity of RNA-primer during DNA reduplication initiation, the telomere ends of somatic cells chromosomes are shortened with every cycle. As a result, after a certain number of doublings, the telomere end is depleted and divisions are terminated due to chromosome erosion (Itahana et al., 2004). This mechanism was confirmed by numerous empirical facts: 90–95% of potentially immortal cancer cells possess telomerase activity and the telomere end of their chromosomes is not shortened; suppression of telomerase activity in these cells causes shortening of the telomere end and division termination, i.e., aging; and restoration of telomerase activity makes them potentially immortal again. Therewith, facts contradictory to this conception were accumulated. The most convincing of them were obtained by a research group led by Blasco (Blasco et al., 1997). They obtained mice zygotes lacking a telomerase gene but with full-sized initial chromosome telomere ends. Mice developed from these zygotes were not only viable, but also fertile. This initial telomere length was sufficient to maintain normal viability of six mouse generations. In the first generation, for example, mice passed through youth and maturity successfully and died in old age having 80% of telomeres in reserve. Only in the fifth and sixth generations did anomalies caused by chromosome telomere end depletion appear. These data were confirmed by another group of authors led by Herrera (Herrera et al., 1999). They obtained an analogous mouse line, but with a shortened initial telomere end, and repeated the experiments of Blasco et al. These mice were viable for only four generations, and anomalies in late generations were related with depletion of telomeres in cells of tissues with the most intensive proliferation (Lee et al., 1998). By the present time, researchers of the telomere mechanism incline to the conclusion that loss of the telomere end indeed leads to chromosome erosion and cell death, but cell proliferation termination during normal physiological cell aging happens earlier than this critical moment and a cell that has expended all its proliferative potential still contains a significant telomere reserve. The telomere mechanism serves as an additional barrier on the road to reproduction of malignant cells (Itahana et al., 2004). The conclusion that there is nonparticipation of the telomere apparatus in the mechanism of termination of old cells' division could have been drawn from the very beginning. It followed from the results of the initial Hayflick experiments that, after a certain number of doublings, a cell enters a nondividing, but viable, state, and there is no sense in discussing viability if division termination due to chromosome

Itahana et al., 2004; Yegorov & Zelenin, 2003).

**4.2 Modern views on the cause of cell proliferation limitation** 

erosion is accepted. Therefore, the question of the Hayflick limit's nature is without answer. Apparently, an alternative reason for this phenomenon should be looked for in the mechanism of cell division.
