Expression of the mitochondrial creatine kinase genes

Mitochondrial Creatine Kinase (MtCK) is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier, creatine, and exists in mammals as two isoenzymes encoded by separate genes. In rats and humans, sarcomere-specific MtCK (sMtCK) is expressed only in skeletal and heart muscle, and has 87% nucleotide identity across the 1257 bp coding region. The ubiquitous isoenzyme of MtCK (uMtCK) is expressed in many tissues with highest levels in brain, gut, and kidney, and has 92% nucleotide identity between the 1254 bp coding regions of rat and human. Both genes are highly regulated developmentally in a tissue-specific manner. There is virtually no expression of sMtCK mRNA prior to birth. Unlike cytosolic muscle CK (MCK) and brain CK (BCK), there is no developmental isoenzyme switch between the MtCKs. Cell culture models representing the tissue-specific expression of either sMtCK or uMtCK are available, but there are no adequate developmental models to examine their regulation. Several animal models are available to examine the coordinate regulation of the CK gene family and include 1) Cardiac Stress by coarctation (sMtCK, BCK, and MCK), 2) Uterus and placenta during pregnancy (uMtCK and BCK), and 3) Diabetes and mitochondrial myopathy (sMtCK, BCK, and MCK). We report the details of these findings, and discuss the coordinate regulation of the genes necessary for high-energy transduction.     

Crystal structure of human ubiquitous mitochondrial creatine kinase

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.     

Divergent enzyme kinetics and structural properties of the two human mitochondrial creatine kinase isoenzymes

The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding

Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degrees C, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (2000) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications.     

Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation,octamer destabilization,and identification of involved residues

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.     

Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.