Thomas M. Kriegel

Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.

The Unique Hexokinase of Kluyveromyces lactis MOLECULAR AND FUNCTIONAL CHARACTERIZATION AND EVALUATION OF A ROLE IN GLUCOSE SIGNALING

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.

Interaction of murine monoclonal subunit-specific antibodies with phosphofructokinase- 1 from Saccharomyces cerevisiae**

Murine monoclonal subunit‐specific antibodies against heterooctameric phosphofructokinase‐1 from Saccharomyces cerevisiae exhibiting no cross‐reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS‐denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteolytic degradation. Strong complex formation was found with one α‐specific antibody. Western blotting employing different enzyme forms allowed to localize epitope sites of the α‐polypeptide chain. Monoclonal antibodies protect phosphofructokinase‐1 against chymotryptic degradation.

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis: A MOLECULAR BASIS FOR UNDERSTANDING THE CONTROL OF YEAST HEXOKINASE FUNCTIONS VIA COVALENT MODIFICATION AND OLIGOMERIZATION.

Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases.

Interaction of 6-phosphofructokinase with cytosolic proteins of Saccharomyces cerevisiae

Hetero‐octameric 6‐phosphofructokinase (Pfk‐1) from Saccharomyces cerevisiae is composed of two types of subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. Pfk single deletion mutants expressing only one type of subunit exhibit Pfk‐1 activity in vivo which, however, is completely lost immediately after cell disruption. In order to elucidate the preconditions of the in vivo activity of the mutant enzymes composed of either α‐ or β‐subunits, we have investigated their potential interaction with selected heat shock and cytoskeletal proteins, employing co‐immunoprecipitation and immunofluorescence microscopy. Western blot analysis identified the mitochondrial chaperonin Hsp60, as well as the cytoskeleton proteins α‐tubulin and actin, in complexes with Pfk‐1 that were co‐precipitated from a cell‐free extract of a pfk2 single deletion mutant expressing only the α‐subunit. The interaction of the corresponding mutant enzyme and Hsp60 was found to depend on the ATP concentration of the extract. Immunofluorescence microscopy displayed a conspicuously filamentous arrangement of the Pfk‐1 mutant protein, exclusively in the pfk2 single deletion mutant. The analysis of structure and activity of Pfk‐1 expressed in S. cerevisiae mutant strains defective in various heat shock proteins (TRiC/CCT, Hsp70, Hsp 104) and in the respective wild‐type background did not reveal significant differences. Copyright

Saccharomyces cerevisiae gene YMR291W/TDA1 mediates the in vivo phosphorylation of hexokinase isoenzyme 2 at serine-15.

Hxk2 is the predominant hexokinase of Saccharomyces cerevisiae during growth on glucose. In addition to its role in glycolysis, the enzyme is involved in glucose sensing and regulation of gene expression. Glucose limitation causes the phosphorylation of Hxk2 at serine‐15 which affects the nucleo‐cytoplasmic distribution and dimer stability of the enzyme. In order to identify the responsible kinase, we screened selected protein kinase single‐gene deletion mutants by high resolution clear native PAGE. Deletion of YMR291W/TDA1 resulted in the absence of the Hxk2 phosphomonomer, indicating an indispensable role of the corresponding protein in Hxk2 phosphorylation.

Identification and characterization of CaApe2 – a neutral arginine/alanine/leucine-specific metallo-aminopeptidase from Candida albicans

The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2, which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. Taking into account the results of DNA and protein sequence analysis including inspection of the genomic region upstream of ORF CaO19.5197, the gene CaAPE2 is likely to consist of two exons linked by a phase-2 intron with exons 1 and 2 encoding a signal peptide and the amino acids 88-954 of ORF CaO19.5197, respectively. The isolated CaApe2 protein shares an equally high similarity with the gene products ScAap1 and ScApe2, suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role that secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans.

Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.

Background: Monomer-dimer equilibrium, substrate affinity, and subcellular localization of yeast hexokinase ScHxk2 depend on the state of phosphorylation of serine 15. Results: Serine/threonine protein kinase Ymr291w/Tda1 is essentially required for ScHxk2-S15 phosphorylation. Conclusion: Ymr291w/Tda1 is the ScHxk2-S15 kinase or an upstream regulatory enzyme. Significance: The analysis of Ymr291w/Tda1 function(s) is indispensable for understanding glucose signaling in yeast. The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.

Studies on triazine dye‐enzyme interaction by means of affinity partitioning

Reactive dyes have found widespread interest as biomimetic ligands in numerous subjects of enzymology and protein chemistry, taking advantage of the principle of biorecognition. The use of Cibacron Blue F3G-A and other triazine dyes as ligands in affinity chromatography and in related techniques has been successfully demonstrated in diverse isolation procedures of enzymes and proteins. In the last few years affinity partitioning became attractive for studying dye-protein interaction because of its high sensitivity in the recognition of ligand-protein binding and of its methodical simplicity.

Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization.