Identification and characterization of the mitochondrial targeting sequence and mechanism in human citrate synthase

Citrate synthase (CS), the first and rate‐limiting enzyme of the tricarboxylic acid (TCA) cycle, plays a decisive role in regulating energy generation of mitochondrial respiration. Most mitochondrial proteins are synthesized in the cytoplasm as preproteins with an amino (N)‐terminal mitochondrial targeting sequence (MTS) that directs mitochondria‐specific sorting of the preprotein. However, the MTS and targeting mechanism of the human CS protein are not fully characterized. The human CS gene is a single nuclear gene which transcribes into two mRNA variants, isoform a (CSa) and b (CSb), by alternative splicing of exon 2. CSa encodes 466 amino acids, including a putative N‐terminal MTS, while CSb expresses 400 residues with a shorter N terminus, lacking the MTS. Our results indicated that CSa is localized in the mitochondria and the N‐terminal 27 amino acids, including a well‐conserved RXY ↓ (S/A) motif (the RHAS sequence), can efficiently target the enhanced green fluorescent protein (EGFP) into the mitochondria. Furthermore, site‐directed mutagenesis analysis of the conserved basic amino acids and serine/threonine residues revealed that the R9 residue is essential but all serine/threonine residues are dispensable in the mitochondrial targeting function. Moreover, RNA interference (RNAi)‐mediated gene silencing of the preprotein import receptors, including TOM20, TOM22, and TOM70, showed that all three preprotein import receptors are required for transporting CSa into the mitochondria. In conclusion, we have experimentally identified the mitochondrial targeting sequence of human CSa and elucidated its targeting mechanism. These results provide an important basis for the study of mitochondrial dysfunction due to aberrant CSa trafficking. J. Cell. Biochem. 107: 1002–1015, 2009. © 2009 Wiley‐Liss, Inc.     

Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant

This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution ¹³C NMR. The oxidation of [1,2,3-¹³C]propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS^?) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS). The ¹³C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the ¹³C spectra of cells presented with [3-¹³C] propionate or [2-¹³C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.     

Activation by spermine of citrate synthase from porcine heart

Spermine activated citrate synthase from porcine heart by decreasing the Km value for the substrate oxaloacetate without affecting the maximal velocity. Spermine markedly increased the maximal velocity of the saturation function with respect to acetyl-CoA as the substrate under conditions of intracellular concentrations of oxaloacetate, but the enzyme was not activated by spermine under conditions of higher concentrations of oxaloacetate. The concentration of spermine required for 50% activation of the enzyme was about 50 microM. Spermidine showed only a little activation, while putrescine caused no activation. Spermine, which contributes to an activation of Ca2(+)-sensitive dehydrogenases of the citric acid cycle by enhancing Ca2+ uptake into mitochondria, can activate citrate synthase directly, and is responsible for the stimulation of oxidative metabolism in mitochondria.     

Controls of citrate synthase activity

The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an $\text{in vitro}$ catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration $\text{in vivo}$ compared to its concentration $\text{in vitro}$. In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available $\text{in vitro}$ so that little can be said concerning the $\text{in situ}$ control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes $\text{in vitro}$ can only serve as a guideline for parameters to study when techniques are available to study control of enzymes $\text{in situ}$.     

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.     

Studies on yeast peroxisomal citrate synthase

Peroxisomal (nonmitochondrial) citrate synthase (CS2) has been purified from a Saccharomyces cerevisiae strain in which the gene for the mitochondrial citrate synthase (CS1) had been disrupted and no CS1 protein is produced. The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes.     

Induction of citrate synthase by aldosterone in the rat kidney

Summary The possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 g/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theK _m s for acetyl-CoA and oxalacetate but augmentedV _max of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 g/100 g body wt) or the diluent, and simultaneously with³H or³⁵S methionine (500 Ci/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 g/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either³H-or³⁵S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 g/100 g body wt) or spirolactone (SC-26304) (80 g/100 g body wt). An equimolar dose of dexamethasone (0.8 g/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.     

Interaction between citrate synthase and thiolase

Thiolase, a mitochondrial matrix enzyme which produces CoASAc from fatty acids, is shown to interact with citrate synthase, the mitochondrial matrix enzyme responsible for CoASAc utilization. The interaction is demonstrated in three ways: the two enzymes co-precipitate in polyethylene glycol; thiolase causes a change in the fluorescence anisotropy of labeled citrate synthase; and the two enzymes co-elute in gel permeation chromatography. The interactions are shown to be specific by the use of enzymes not metabolically related to citrate synthase.     

Reactivation of denatured citrate synthase.

1. The imported mitochondrial enzyme citrate synthase can be partially (less than or equal to 45%) reactivated after denaturation in guanidinium chloride, if the concentration of the denaturing agent is lowered by dialysis, rather than by dilution, when essentially no reactivation is observed. 2. The presence of a reducing agent (dithiothreitol) is necessary for regain of activity. 3. Optimum regain of activity occurs at enzyme concentrations of about 10-20 micrograms/ml; at higher concentrations there is significant formation of aggregates.     

Melatonin: A promising agent targeting leukemia

Leukemia or cancer of blood is a well-known cancer, which affects a range of people from newborns to the very old. It is a public health problem throughout the world. By way of treatment, due to the lack of specific anticancer therapies, common treatments of leukemia lead to severe side effects. Nonspecific anticancer drugs result in inhibition of normal cell growth and thereby their necrosis. Moreover, drug resistance is an additional problem, which stands in the way of leukemia treatment. Thus, finding new treatments for leukemia is essential. Melatonin, as a natural product, has been shown to be effective in a wide variety of diseases such as coronary heart disease, schizophrenia, chronic pain, and Alzheimer's disease. In addition, melatonin levels have been observed to be altered in different cancers, such as breast cancer, colorectal cancer endometrial cancer, and hematopoetical cancers. Anticancer features of melatonin such as pro-oxidation, apoptosis induction, antiangiogenesis property and metastasis and invasion inhibition suggest that this natural compound can be used as a potential agent in novel therapeutic strategies for cancers. Also, it has been reported that melatonin has positive and protective effects on different physiological reactions and in normal bone marrow cells suggesting effectiveness in leukemia therapy. Thus, the aim of our paper was to depict and summarize the main molecular targets of melatonin on leukemia models.