Tài liệu Báo cáo khoa học: Changes in rat liver mitochondria with aging Lon protease-like activity

Changes in rat liver mitochondria with aging

Lon protease-like activity and Ne

-carboxymethyllysine accumulation in the matrix

Hilaire Bakala1

, Evelyne Delaval1

, Maud Hamelin1

, Jeanne Bismuth1

, Caroline Borot-Laloi1

,

Bruno Corman2 and Bertrand Friguet1

1

Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite´ Paris7-Denis Diderot, Paris, France; 2

Service de Biologie Cellulaire, Commissariat a` l’Energie Atomique/Saclay, Gif-sur-Yvette, France

Aging is accompanied by a gradual deterioration of cell

functions. Mitochondrial dysfunction and accumulation of

protein damage have been proposed to contribute to this

process. The present study was carried out to examine the

effects of aging in mitochondrial matrix isolated from rat

liver. The activity of Lon protease, an enzyme implicated in

the degradation of abnormal matrix proteins, was meas￾ured and the accumulation of oxidation and glycoxidation

(Ne

-carboxymethyllysine, CML) products was monitored

using immunochemical assays. The function of isolated

mitochondria was assessed by measuring respiratory chain

activity. Mitochondria from aged (27 months) rats exhi￾bited the same rate of oxygen consumption as those from

adult (10 months) rats without any change in coupling

efficiency. At the same time, the ATP-stimulated Lon

protease activity, measured as fluorescent peptides released,

markedly decreased from 10-month-old rats (1.15 ±

0.15 FUÆlg protein)1

Æh)1

) to 27-month-old-rats (0.59 ±

0.08 FUÆlg protein)1

Æh)1

). In parallel with this decrease in

activity, oxidized proteins accumulated in the matrix upon

aging while the CML-modified protein content assessed

by ELISA significantly increased by 52% from 10 months

(11.71 ± 0.61 pmol CMLÆlg protein)1

) to 27 months

(17.81 ± 1.83 pmol CMLÆlg protein)1

). These results

indicate that the accumulation of deleterious oxidized and

carboxymethylated proteins in the matrix concomitant

with loss of the Lon protease activity may affect the ability

of aging mitochondria to respond to additional stress.

Keywords: aging; mitochondria; matrix; Lon protease;

carboxymethyllysine.

A striking characteristic of normal aging in long-lived

animals is the gradual decline in their physiological func￾tions. This decline is associated with an increase in reactive

oxygen species (ROS) production [1] and an accumulation

of macromolecules damaged by post-translational nonenzy￾matic modifications which alter the structure and function

of tissue and cellular proteins [2–5]. Under oxidative stress,

carbohydrates, lipids and proteins are the major targets of

reactive oxygen species. Proteins can be damaged either

directly or indirectly through the reactive carbonyl com￾pounds derived from the oxidation of carbohydrates and

lipids [6,7]. These carbonyl compounds react with protein

amino-groups to give glycoxidation products such as

Ne

-carboxymethyllysine (CML) [8]. This glycoxidation

process modifies cell proteins and the cumulative effects

lead to the tissue alterations and cell dysfunction typical of

aging and diabetes [9,10]. Recent data also indicate that

glycoxidative processes affect mitochondrial membrane

phospholipids [11]. Mitochondria are in fact a major

intracellular source of ROS during oxidative phosphoryla￾tion and the increased production of ROS is implicated in

the aging process [12–16]. Mitochondria are also the major

targets of these ROS, which may damage mitochondrial

proteins themselves, leading to dysfunction of these

organelles [17,18]. Oxidative damage mainly concerns

the activities of electron transport complexes of the inner

mitochondrial membrane which are specifically modified

during aging [19–21].

No attempt has yet been made to correlate the alterations

in mitochondrial function with biochemical changes in the

liver mitochondrial matrix of aging rats. Nevertheless, an

age-related decrease in the expression of several genes

involved in mitochondrial bioenergetics and mitochondrial

biogenesis occurs in aging mice; the largest age-associated

alteration affects the matrix enzyme Lon protease [22]. This

ATP-stimulated protease is homologous to bacterial Lon

protease [23] and has been found in several mammals

including human tissues and cell lines [24–26]. It is

responsible for the degradation of abnormal proteins and

certain short-lived specific proteins [27,28].

In this study we have investigated the age-associated

biochemical changes in mitochondrial matrix proteins

from the rat liver. For this purpose we related the activity

of Lon protease with the level of damaged proteins in the

matrix, focusing on the accumulation of oxidized and

CML-proteins as a marker of glycoxidative stress.

Correspondence to H. Bakala, Laboratoire de Biologie et Biochimie

Cellulaire du Vieillissement, Universite´ Paris7-Denis Diderot,

T23-33 1er e´tage CC 7128, 2 Place Jussieu, 75252 Paris, France.

Fax: + 33 1 44 27 82 34; Tel.: + 33 1 44 27 82 35;

E-mail: [email protected]

Abbreviations: ROS, reactive oxygen species; AGE, advanced glycat￾ion end products; CML, carboxymethyllysine; ABTS, 2,2¢-azinobis

(3-ethylbenzo-6-thiazolinesulfonic acid); RCR, respiratory

control ratio.

(Received 22 January 2003, accepted 27 March 2003)

Eur. J. Biochem. 270, 2295–2302 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03598.x