guanosine-triphosphate and Cataract

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Biological Transport, Active; Cataract; Cyclic AMP; Diffusion; Fructosephosphates; Glucose; Glucosephosphates; Glycolysis; Guanosine Triphosphate; Hexokinase; Humans; Insulin; Isoenzymes; Kinetics; Lens, Crystalline; Phosphates; Phosphofructokinase-1

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary 'RAB3GAP' complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.

Topics: Abnormalities, Multiple; Animals; Blotting, Western; Case-Control Studies; Cataract; Cells, Cultured; Cornea; Cytosol; Endoplasmic Reticulum; Fibroblasts; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation; Guanosine Triphosphate; HeLa Cells; Humans; Hydrolysis; Hypogonadism; Intellectual Disability; Mice; Mice, Knockout; Microcephaly; Optic Atrophy; rab GTP-Binding Proteins; rab1 GTP-Binding Proteins; rab3 GTP-Binding Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.

Topics: Aging; Animals; Blotting, Western; Calcium; Calcium Chloride; Cataract; Cell Line; Cell Line, Tumor; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Enzyme Activation; GTP-Binding Proteins; Guanosine Triphosphate; HeLa Cells; Humans; Hydrogen Peroxide; K562 Cells; Lens, Crystalline; Mice; NIH 3T3 Cells; Oxidative Stress; Protein Glutamine gamma Glutamyltransferase 2; Time Factors; Transfection; Transglutaminases; Ultraviolet Rays

On account of its protein crosslinking activity, the Ca2+-dependent transglutaminase of the lens is likely to be involved in the formation of cataracts. We have now purified the rabbit lens enzyme to near homogeneity as judged by SDS-PAGE (Mr approximately 78 kDa), and a key feature of the procedure was the use of a highly selective affinity chromatographic step with a fibronectin fragment as ligand. The catalytic activity of the lens transglutaminase, measured by the incorporation of dansylcadaverine into dimethylcasein, was compared with those of two similar enzymes isolated from human red cells and from guinea pig liver, respectively. All three enzymes were inhibited by GTP, but the lens enzyme was most sensitive to inhibition by the nucleotide. Moreover, GTP was also shown to inhibit the formation of the approximately 55 kDa betacrystallin dimers in the Ca2+-treated rabbit lens homogenate, proving that the nucleotide is a negative regulator for the crosslinking activity of transglutaminase in this tissue.

Topics: Animals; Calcium; Catalysis; Cataract; Chromatography, Ion Exchange; Cross-Linking Reagents; Crystallins; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Guanosine Triphosphate; Guinea Pigs; Humans; Lens, Crystalline; Liver; Rabbits; Transglutaminases

Sensitive high-performance liquid chromatography methods were employed to assess regional distribution of adenine, guanosine and uridine nucleotides in clear and cataractous human eye lenses. According to slit-lamp examination, three forms of senile cataract were distinguished: (1) supranuclear or deep cortical cataract (typical senile cataract), (2) primary nuclear cataract (cataracta brunescens) and (3) subcapsular cortical cataract associated either with a supranuclear (3a) or a secondary nuclear cataract (3b). Except for AMP, which was highest in the nuclear fraction, all other nucleotides (ATP, ADP, GTP, and UTP) were predominantly located in the anterior cortex (plus epithelium) of clear as well as cataractous lenses, that is, ATP levels in the nucleus amounted to 20% of those found in the anterior cortex (plus epithelium); ATP levels in the posterior cortex were about 60% of those in the anterior cortex (plus epithelium). Significant differences in the absolute regional nucleotide level existed between the different forms of cataract. Highest ATP levels were found in the anterior cortex (plus epithelium) of clear lenses and deep or supranuclear cortical cataract. The ATP level was slightly diminished in primary nuclear cataract and in supranuclear cortical cataract when associated with an early subcapsular cortical cataract. ATP levels were depressed to less than 30% in the anterior cortex (plus epithelium) of lenses with a subcapsular cortical cataract when associated with either an early secondary nuclear or a mature cataract. Furthermore, the ATP/ADP ratio was decreased in this form of senile cataract. The decrease in lens nucleotide level did not correlate with increased age. These data suggest that decreases in regional ATP level are a secondary event and do not appear to be causally involved in the genesis of the 'cataracta senilis'.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Cataract; Chromatography, High Pressure Liquid; Guanosine Triphosphate; Humans; Lens Cortex, Crystalline; Lens Nucleus, Crystalline; Lens, Crystalline; Nucleotides; Uridine Triphosphate

Topics: Adenosine Triphosphate; Aged; Amino Acids; Animals; Arginine; Aspartic Acid; Biological Transport; Cataract; Crystallins; Guanosine Triphosphate; Humans; Lens, Crystalline; Leucine; Rabbits; Tritium