Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-04
2003-09-16
Fredman, Jeffrey (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023100, C536S023500
Reexamination Certificate
active
06620594
ABSTRACT:
This application is the National Stage of International Application No. PCT/EP98/02645, filed on May 5, 1998.
The present invention relates to a cloned gene which codes for an uncoupling protein (UCP3
L
) which controls thermogenesis in human skeletal muscle and heart.
The present invention also relates to a cloned gene which codes for an uncoupling protein (UCP3
S
), also controlling thermogenesis in human skeletal muscle and heart.
A further aspect of the present invention relates to the use of the said cloned genes for correcting dysfunctions of thermogenesis in human skeletal muscle and heart.
A dysfunction of thermogenesis can induce disorders such as obesity or cachexia.
Obesity is characterized by an excess of adipose mass which can represent more than 30% of the bodyweight. The incidence of this disturbance of energy balance is constantly increasing in industrialized countries. Preventing the development of obesity, or treating it, would enable the complications associated with this pathology, namely cardiovascular diseases, hypertension and type II diabetes, to be avoided.
In some special cases, the control of thermogenesis could also prove useful. In man, weight loss following a slimming diet induces a saving of energy by the body. On resumption of normal feeding, the energy expenditure remains reduced until the body has recovered the adipose mass and the lean mass which it has lost previously. This decrease in energy expenditure often leads to an excess weight regain. A similar problem is encountered in endurance athletes as soon as they stop training. In effect, in a trained person, the energy expenditure is decreased relative to that of a sedentary person. This energy saving is responsible for a substantial weight gain (most especially of fat) after chronic physical activity has been stopped.
Cachexia is a metabolic situation in which the energy expenditure exceeds the food intake. Its main causes are underfeeding (e.g. anorexia nervosa), cancer, infectious diseases including AIDS and a state of shock. The decrease in adipose and muscle masses can threaten the individual's survival.
The energy expenditure is increased in the mitochondria by uncoupling of the oxidative phosphorylations. The oxidations induce the exit of protons (H
+
) from the mitochondrion, creating a proton gradient which, as it dissipates, permits the synthesis of ATP. Uncoupling can be induced by chemical compounds such as 2,4-dinitrophenol (DNP) and by other acidic aromatic compounds. These substances carry H
+
from the outside to the inside of the inner mitochondrial membrane. In the presence of these uncoupling agents, the oxidation of NADH takes place normally, but ATP is not formed by the mitochondrial ATP synthetase since the proton gradient is dissipated.
Brown adipose tissue (BAT), which is very rich in mitochondria, is specialized in this process of thermogenesis. The inner membrane of its mitochondria contains a large amount of an uncoupling protein (UCP) which enables protons to return from the outside to the inside of the mitochondrion. In essence, the uncoupling protein produces heat by short-circuiting the battery of mitochondrial protons. In spite of its tissue specificity, UCP is a member of the mitochondrial carrier family, which includes the ATP/ADP, phosphate and 2-oxoglutarate/malate carriers, in particular. In contrast to the ATP/ADP carrier, which is a constitutive carrier, UCP is subjected to substantial regulatory processes (M. Klingenberg,
J. Bioenerg. Biomembr
. 25, 447 (1993)). Its activity is decreased by purine di- or triphosphate nucleotides and increased by fatty acids (J. Nedergaard, B. Cannon, in
New Comprehensive Biochemistry
(
Bioenergetics
) L. Ernster, Ed (Elsevier Science, Stockholm, 1992), vol. 23, 385).
The uncoupling of oxidative phosphorylation is very useful from a biological standpoint; it is a means for BAT to generate heat in order to maintain a physiological temperature in the newborn offspring of some animals (including man) and in the mammals of the cold regions.
The human UCP gene is localized at 4q31; it consists of six exons and codes for a protein of 307 amino acids with no targeting presequence signal. Like other mitochondrial carriers, UCP is inserted into the mitochondrial membrane by six hydrophobic &agr;-helical domains, each encoded by a portion of the six exons (L. P. Kozak et al.,
J. Biol. Chem
. 263, 12274 (1988)). Its polypeptide chain consists of three related sequences in tandem of approximately 100 amino acids, each encoded by two exons and corresponding to two transmembrane domains (F. Bouillaud et al.,
Biochem. Biophys. Res. Commun
. 157, 783 (1988)). Topological studies suggest that the amino- and carboxy-terminal ends of UCP are oriented towards the cytosol side of the inner mitochondrial membrane (B. Miroux et al.,
EMBO J
. 12, 3739 (1993)). Furthermore, J. Nedergaard et al. (
New Comprehensive Biochemistry
(
Bioenergetics
) L. Ernster, Ed. (Elsevier Science, Stockholm, 1992), vol. 23, 385) and D. Ricquier et al. (
FASEB J
. 5, 2237 (1991)) have shown that the expression of UCP is increased at transcriptional level by noradrenaline. This effect is mediated by stimulation of the three subtypes of &bgr;-adrenoceptors (&bgr;
1
, &bgr;
2
and &bgr;
3
) and of the &agr;
1
-adrenoceptor.
However, M. E. J. Lean et al. (
Brown Adipose Tissue
P. Trayhurn, D. G. Nicholls, Eds, (Edward Arnold, London, 1986), 339) have shown that, in man, the BAT, expressing UCP, present in newborn infants decreases considerably in adults. Hence, under physiological conditions, BAT cannot play an important part in non-shivering thermogenesis in man.
On the other hand, L. Simonsen et al. (
Int. J. Obes
. 17, S47 (1993)) have determined that, in man, skeletal muscle contributes to the extent of approximately 40% to adrenaline-induced body thermogenesis. Studies performed on rats (P. L. Thurlby et al.,
Can. J. Physiol. Pharmacol
. 64, 1111 (1986); I. Nagase et al.,
J. Clin. Invest
. 97, 2898 (1996)) suggest that adrenaline-induced thermogenesis in human skeletal muscle (L. Simonsen et al.,
Int. J. Obes
. 17, S47 (1993)) could be mediated by a muscle UCP.
In the search for homologues of UCP in human skeletal muscle, we have screened a library of human skeletal muscle cDNA, and three clones (UCP2, UCP3
L
and UCP3
S
) have been isolated. Whereas UCP2 mRNA has been found in all human tissues studied, as has also been described by Fleury et al. (
Nature Genet
. 15, 269 (1997)), UCP3 has proved to be very specific to human skeletal muscle. This new member of the UCP family, with a strong specificity of expression in skeletal muscle, is involved in the control of oxidative phosphorylations in man.
The characterization of the genes coding for UCP3
L
and UCP3
S
in terms of nucleotides and of amino acids is described here for the first time.
One aspect of the present invention is hence a DNA fragment, characterized in that it contains the nucleotide sequence depicted in SEQ ID NO 3, the said DNA fragment coding for an uncoupling protein (UCP3
L
) which is characterized by the amino acid sequence depicted in SEQ ID NO 4, or in that it contains a homologous sequence coding for the same amino acid sequence. Preferably, the chosen DNA fragment originates from human skeletal muscle.
Another aspect of the present invention is a DNA fragment, characterized in that it contains the nucleotide sequence depicted in SEQ ID NO 5, the said DNA fragment coding for an uncoupling protein (UCP3
S
) possessing the amino acid sequence depicted in SEQ ID NO 6, or in that it contains a homologous sequence coding for the same amino acid sequence. Preferably, the chosen DNA fragment originates from human skeletal muscle.
One subject of the present invention is an uncoupling protein, characterized in that it comprises the amino acid sequence depicted in SEQ ID NO 4.
Another subject of the present invention is an uncoupling protein, characterized in that it comprises the amino acid sequence depicted in SEQ ID NO 6.
Furthermore, the present invention sets out to
Boss Olivier
Giacobino Jean-Paul
Muzzin Patrick
Ferraro Gregory D.
Fredman Jeffrey
Kaushai Samesh
Novartis AG
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