Differential Expression Of The Protein SUMO Enzymes In Normal Eye Tissues And Models Of Major Eye Diseases

The vertebrate eye is a unique organ which consists of cornea,lens and retina and can visualize the outside world.The developmental abnormality of the ocular tissues will cause severe pathogenesis of major ocular diseases.Cataract is one of the most blindness-causing diseases.Its major phenotype is the development of opacity,and eventual loss of vision.It has been shown that non-congenital cataract is derived from free radical damage,induced apoptosis of lens epithelial cells,induced damage of lens proteins and aging.The current treatment of cataract is surgical operation to replace the cataract lens with a transparent artificial lens.This procedure,however,has numerous shortcomings such as development of secondary cataract and blare vision etc.Age-related macular degeneration(AMD),another vision-threatening ocular disease,is usually observed in elderly people,and caused by both genetic and environmental factors.The major characteristics of AMD are the loss of first retinal pigmental epithelial cells followed by the loss of photoreceptors due to triggering of apoptosis.According to the presence or absence of vascular development,AMD can be categorized as wet AMD(with vascular development)and dry AMD.While anti-VEGF has been traditionally used to treat wet AMD,there is no therapeutic strategy for dry AMD.Thus,studies on the molecular mechanisms mediating pathogenesis of cataract and AMD,and development of effective treatment strategy for the above ocular diseases are fundamental issues in improving human health.In this regard,the present thesis employs the animal models of both cataract and AMD to investigate the possible involvement of sumoylation enzymes in these major ocular diseases.Protein sumoylation refers to the biochemical reaction in which a small ubiquitin-like modifiers,(SUMOs for short)is added to the target protein.It is an important post-translational modification,and has very important physiological functions,especially in regulating gene expression at the transcriptional level.Sumoylation is a reversible enzymatic reaction catalyzed by E1 activating enzymes(SAE1 and UBA2),E2 conjugating enzyme UBC9,and several E3 ligases in the presence of ATP as energy supply.During eye development,sumoylation plays a vital role in regulating development of both eye lens and retina.Our lab has previously shown that SUMO 1-conjugated sumoylation is necessary to activate the function of the p32 Pax6,which plays a fundamental role in establishing the lens placode and vesicle.At the same,our work has demonstrated that the activating protein 1(SP1)when conjugated with SUMO1 or SUM02/3,displays contrast functions.While SUMO 1-conjugation promotes lens development,sumoylation of SP1 by SUM02/3 inhibits the differentiation process.In addition,our recent work has shown that SUMO1-mediation p53 sumoylation regulates the transcription of the downstream genes when triggered by the oxidative stress,leading to protection of retina pigment epithelial cells by heterochromatin.Throughout these studies,however,it remains to be determined what ligases are involved in the above sumoylation reactions?What are the expression patterns of the major sumoylation enzyme systems in the eye?What are the changes of the sumoylation enzyme system during cataractogenesis and AMD development?To answer these questions,the present thesis analyzed the normal expression patterns of the 5 major sumoylation enzymes:SAE1,UBA2,UBC9,PIAS1 and RanBP2 in major ocular tissues and common ocular cell lines:αTN4-1,HLE,FHL124,N/N1003A and ARPE-19,and established animal models for both cataract and AMD,and examined the expression pattern changes of the 5 sumoylation enzymes in the in vitro organ cultured cataract models and injected AMD animal models.The first group of experiments in this thesis were to determine the normal expression patterns of both mRNA and protein for SAE1,UBA2,UBC9,PIAS1 and RanBP2 in cornea,lens epithelial cells(LEC),lens fiber cells(LF),and retina of adult mouse eyes.It was found that the mRNAs for SAE1,PIAS1 and RanBP2 display obviously differential expression.At the protein levels,the above 5 enzymes are all expressed in cornea,lens epithelial cells and retina.In the retina,higher level of SAE1,UBA2 and PIAS1 were detected than in other ocular tissues.In contrast,the cornea displayed the highest level of RanBP2 among the major ocular tissues examined.The above 5 sumoylation enzymes were also analyzed in five ocular cell lines including αTN4-1,HLE,FHL124,N/N1003A and ARPE-19.All enzymes were expressed in these cell lines in a relatively similar level.The localization of the 5 enzymes was also examined using immunocytochemistry.Subsequently,in vitro organ culture of cataract models were induced using glucose oxidase(GO)treatment or UVA irradiation.In cataract models,both GO treatment and UVA irradiation caused downregulation of the above enzymes at protein level.However,while GO treatment significantly down-regulates the mJRNAs for all enzymes,UVA slightly up-regulates the mRNAs for the same enzymes.At the same time,mouse models injected with NaIO3 were also established.In the NaIO3-induced AMD,both mRNAs and proteins for the 5 enzymes were downregulated.This downregulation was partially reversed after the treatment of the 2nd day.Clear downregulation of both mRNAs and proteins for SAE1,UBA2,UBC9 and PIAS1 were observed after 3-day treatment.For RanBP2,however,tremendous upregulation was observed at both mRNA and protein level,suggesting that RanPB2 may have non-sumoylation functions.Finally,experiments desired to study the functional mechanisms of PIAS1 were conducted.It was found that PIAS1 was downregulated by GO in a dose-dependent manner.By altering the level of PIAS1 in the αTN4-1 mouse lens epithelial cells using either Cas9-mediated silencing technology or pEGFP-C3-driven overexpression,it was demonstrated PIAS1 can negatively regulate viability of mouse lens epithelial cells.These observations are consistent with previous study in U202 cells,MEF cells and Hela Cells.In summary,using modern molecular and cellular biology technology,the present thesis has determined the expression patterns of the sumoylation enzymes SAE1,UBA2,UBC9,PIAS1 and RanBP2 in major ocular tissues of normal adult,in commonly used ocular cell lines and cataract and AMD eye disease models,and explored the function of PIAS1 in regulating cell viability.These results will lay down a necessary biological foundation for the further study of sumoylation functions in regulating eye development and mediating major ocular diseases.