Carnitine carrier related protein-1

Details Carnitine carrier related protein-1 Carnitine carrier related protein-1

2000-06-21

2004-09-07

Horlick, Kenneth R. (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S252300, C435S320100, C435S325000, C514S04400A, C530S300000, C536S023100

Reexamination Certificate

active

06787647

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to polynucleotides encoding the Carnitine Carrier Related Protein-1 (CCRP-1) protein, fragments thereof, and the regulatory regions located at the 5′- and 3′-end of the CCRP-1 gene. The invention also concerns polypeptides encoded by the CCRP-1 gene and fragments thereof. The invention is further directed to methods of making said polynucleotides and polypeptides as well as methods of using the same. The invention also relates to antibodies directed specifically against the above polypeptides and to methods of using said antibodies to purify and detect the above polypeptides and to detect mitochondrion organelles.
BACKGROUND OF THE INVENTION
The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM; previously designated MOM; Pfanner, N. et al., 1996) and at least three inner membrane proteins which comprise the translocase of inner mitochondrial membrane (TIM; previously designated MIM; Pfanner et al, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import. Preproteins are recognized by surface receptor components of the TOM complex and are translocated through a proteinaceous pore formed by other TOM components. Proteins targeted to the matrix are then recognized by the import machinery of the TIM complex. The import systems of the outer and inner membranes can function independently (Segui-Real, B. et al., 1993). Three TIM proteins have been identified in the yeast Saccharomyces cerevisiae. TIM44 is a hydrophilic protein which is peripherally associated with the inner face of the inner mitochondrial membrane. TIM23 and TIM17 are integral membrane proteins which are thought to comprise the core subunits of the inner membrane translocation channel. (Bomer, U. et al., 1996). Depletion of TIM17 (also known as MIM17, Mpi2, and Sms1; Pfanner et al., supra) causes defects in the import of several mitochondrial proteins (Ryan, K. R. et al., 1994). Furthermore, TIM44, TIM23, and TIM17 proteins are among the few known proteins essential for yeast viability (Maarse, A. C. et al. 1994; Ryan et al., supra).
Fatty acids are activated on the outer mitochondria membrane, whereas they are oxidized in the mitochondria matrix. Long chain acyl CoA molecules do not readily traverse the inner mitochondrial membrane, and so a special transport mechanism is needed. Activated long-chain fatty acids are carried across the inner mitochondrial membrane by carnitine zwitterionic compound formed from lysine. The acyl group is transferred from the sulfur atom of CoA to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyltransferase I, which is located on the cytosolic face of the inner mitochondrial membrane. Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase. The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitine acyltransferase II, is thermodynamically feasible because the O-acyl link in carnitine has a high group-transfer potential. Finally, carnitine is returned to the cytosolic side by the translocase, in exchange for an incoming acylcarnitine. A defect in the transferase or translocase, or a deficiency of carnitine, might be expected to impair the oxidation of long-chain fatty acids.
Uncoupling proteins, such as UCP-1 (thermogenin), are transmembrane proton-translocating proteins present in the mitochondria of brown adipose tissue, a specialized tissue which functions in heat generation and energy balance (Nicolls, D. G., and Locke, R. M., 1984; Rothwell, N. J. and Stock, M. J. 1979). Mitochondrial oxidation of substrates is accompanied by proton transport out of the mitochondrial matrix, creating a transmembrane proton gradient. Re-entry of protons into the matrix via ATP synthase is coupled to ATP synthesis. However, UCP-1 functions as a transmembrane proton transporter, permitting re-entry of protons into the mitochondrial matrix unaccompanied by ATP synthesis. Environmental exposure to cold evokes neural and hormonal stimulation of brown adipose tissue, which increases UCP mediated proton transport, brown fat metabolic activity, and heat production.
Recent studies with transgenic models indicate that brown fat and UCP-1 have an important role in energy expenditure in rodents. Transgenic mice in which brown adipocyte tissue was ablated by a toxin coupled to the UCP-promoter developed obesity and diabetes (Lowell, B. B., et al., 1993). Obesity in these transgenic animals developed in the absence of hyperphagia, suggesting that the uncoupled mitochondrial respiration of brown fat is an important component of energy expenditure. In a separate transgenic mouse model, ectopic expression of UCP-1 in white adipose tissue of genetically-obese mice led to a significant reduction in body weight and fat stores (Kopecky J., et al. 1995). These studies indicate that activity of UCP-1 is accompanied by energy expenditure and weight loss in rodents. Two other UCP proteins have recently been cloned. The first uncoupling protein-like protein (UCPL) or UCP-2, is expressed in multiple tissues, and is enriched in tissues of the lymphoid lineage (Fleury, C., et al., 1997). The second, UCP-3, is predominantly localized to skeletal muscle (Boss, O., et al., 1997). UCP-3 has been found to be regulated by cold and thyroid hormone (Larkin, S., et al., 1997).
Thermogenic protein activity, such as that found with UCP-1, may be useful in reducing, or preventing the development of excess adipose tissue, such as that found in obesity. Obesity is becoming increasingly prevalent in developed societies. Attempts to reduce food intake, or to decrease hypernutrition, are usually fruitless in the medium term because the weight loss induced by dieting results in both increased appetite and decreased energy expenditure (Leibel et al. 1995). The intensity of physical exercise required to expend enough energy to materially lose adipose mass is too great for many obese people to undertake on a sufficiently frequent basis. Thus, obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. In addition obesity carries a serious risk of co-morbities including, Type 2 diabetes, increased cardiac risk, hypertension, atherosclerosis, degenerative arthritis, and increased incidence of complications of surgery involving general anesthesia.
SUMMARY OF THE INVENTION
The present invention provides isolated CCRP-1 polynucleotides and polypeptides. One aspect of the invention provides isolated nucleic acid molecules comprising or alternatively consisting of polynucleotides having a nucleotide sequence selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1; (b) a polynucleotide comprising the nucleotide sequence of the human cDNA contained in the deposited clone; (c) a polynucleotide comprising a portion of the nucleotide sequence of SEQ ID NO:1 coding for a mature CCRP-1 polypeptide; (d) a polynucleotide comprising a nucleotide sequence of the portion of the human cDNA contained in the deposited clone coding for a mature CCRP-1 polypeptide; (e) a polynucleotide comprising a nucleotide sequence coding for the amino acid sequence of the full length polypeptide of SEQ ID NO:2; (f) a polynucleotide comprising a nucleotide sequence coding for an amino acid sequence of a mature polypeptide of SEQ IDNO:2; (g) a polynucleotide comprising a nucleotide sequence coding for an amino acid sequence of a full length CCRP-1 polypeptide encoded by the human cDNA contained in the deposited clone

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