Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients' sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.

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.

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, ␤-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. Male fertility is compromised by the hormonal and metabolic changes that are associated with type-1 and type-2 diabetes (1, 2), obesity (3), and the metabolic syndrome, the latter disturbance sharing essential pathologic features with the former diseases (4). The deleterious influence of diabetes and obesity on fertility is receiving increasing attention because their prevalence and incidence is escalating worldwide, whereas the age at first diagnosis of both diseases is continuously declining (5, 6). Because of this situation, the fertility of a growing number of individuals is affected before and during their reproductive years (7,8). Recently, it was shown in a large cohort study that BMI affects fertility potential at the critical age for reproduction, whereas age had a more dominant effect on ...

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 ringshaped 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. The enzymes of the hexokinase family catalyze the intracellular trapping and the initiation of metabolism of glucose, fructose, and mannose. In addition to their role in glycolysis, an increasing number of yeast, plant, and mammalian hexokinases have been found to represent multifunctional proteins that are implicated in glucose sensing and signaling (1-4), whereas their glycolytic sugar substrate plays a dual role as a carbon source and hormone-like regulator (4, 5). The molecular basis underlying the involvement of hexokinases in the transcriptional control of glucose metabolism and in glucose homeostasis is their ability to interact with mitochondria and to reversibly translocate to nuclei (3, 6 -9).In the Crabtree-positive yeast Saccharomyces cerevisiae, glucose abundance is accompanied by the translocation of the cytosolic hexokinase isoenzyme 2 (ScHxk2) 4 and the transcriptional repressor Mig1 (ScMig1) into the nucleus, where both proteins participate in the formation of a hetero-oligomeric complex that suppresses the transcription of ScMig1 target genes like SUC2 encoding invertase (9). The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).Contrary to the situation in bakers' yeast, glucose and fructose limitation causes the translocation of the mammalian hexokinase isoenzyme IV (also referred to as "glucokinase" or hexokinase D) to the nucleus of the liver parenchymal cell where it binds to its regulatory protein, GKRP, and retranslocates when glucose is abundantly av...

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.