HSP90ß prevents aging-related cataract formation through regulation of the charged multivesicular body protein (CHMP4B) and p53.

Cataract is a leading ocular disease causing global blindness. The mechanism of cataractogenesis has not been well defined. Here, we demonstrate that the heat shock protein 90ß (HSP90ß) plays a fundamental role in suppressing cataractogenesis. HSP90ß is the most dominant HSP in normal lens, and its constitutive high level of expression is largely derived from regulation by Sp1 family transcription factors. More importantly, HSP90ß is significantly down-regulated in human cataract patients and in aging mouse lenses, whereas HSP90ß silencing in zebrafish causes cataractogenesis, which can only be rescued by itself but not other HSP90 genes. Mechanistically, HSP90ß can directly interact with CHMP4B, a newly-found client protein involved in control of cytokinesis. HSP90ß silencing causes upregulation of CHMP4B and another client protein, the tumor suppressor p53. CHMP4B upregulation or overexpression induces excessive division of lens epithelial cells without proper differentiation. As a result, these cells were triggered to undergo apoptosis due to activation of the p53/Bak-Bim pathway, leading to cataractogenesis and microphthalmia. Silence of both HSP90ß and CHMP4B restored normal phenotype of zebrafish eye. Together, our results reveal that HSP90ß is a critical inhibitor of cataractogenesis through negative regulation of CHMP4B and the p53-Bak/Bim pathway.

WRKY75 regulates anthocyanin accumulation in juvenile citrus tissues.

The anthocyanin accumulation in juvenile tissues can enhance the ornamental value, attract pollinators, and help improve abiotic stress. Although transcriptional regulation studies of anthocyanin have been relatively extensive, there are few reports on the mechanism of anthocyanin accumulation in young tissues. This study reveals that many juvenile citrus tissues (flowers, leaves, and pericarp) undergo transient accumulation of anthocyanins, exhibiting a red coloration. Using weighted gene co-expression network analysis (WGCNA) identified CitWRKY75 as a candidate gene. After detecting the expression levels of CitWRKY75 in various citrus juvenile tissues, the expression trend of CitWRKY75 was highly consistent with the red exhibiting and fading. Overexpression of CitWRKY75 in tobacco significantly increased the anthocyanin content. LUC and yeast one-hybrid assay demonstrated that CitWRKY75 could bind to the promoter of CitRuby1(encoding the key transcription factor promoting anthocyanin accumulation) and promote its expression. Finally, comparing the expression levels of CitWRKY75 and CitRuby1 in the late development stage of blood orange found that CitWRKY75 was not the main regulatory factor for anthocyanin accumulation in the later stage. This study used reverse genetics to identify a transcription factor, CitWRKY75, upstream of CitRuby1, which promotes anthocyanin accumulation in citrus juvenile tissues. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01490-9.

A transcriptional cascade involving BBX22 and HY5 finely regulates both plant height and fruit pigmentation in citrus.

Dwarfing is a pivotal agronomic trait affecting both yield and quality. Citrus species exhibit substantial variation in plant height, among which internode length is a core element. However, the molecular mechanism governing internode elongation remains unclear. Here, we unveiled that the transcriptional cascade consisting of B-BOX DOMAIN PROTEIN 22 (BBX22) and ELONGATED HYPOCOTYL 5 (HY5) finely tunes plant height and internode elongation in citrus. Loss-of-function mutations of BBX22 in an early-flowering citrus (Citrus hindsii "SJG") promoted internode elongation and reduced pigment accumulation, whereas ectopic expression of BBX22 in SJG, sweet orange (C. sinensis), pomelo (C. maxima) or heterologous expression of BBX22 in tomato (Solanum lycopersicum) significantly decreased internode length. Furthermore, exogenous application of gibberellin A3 (GA3) rescued the shortened internode and dwarf phenotype caused by BBX22 overexpression. Additional experiments revealed that BBX22 played a dual role in regulation internode elongation and pigmentation in citrus. On the one hand, it directly bound to and activated the expression of HY5, GA metabolism gene (GA2 OXIDASE 8, GA2ox8), carotenoid biosynthesis gene (PHYTOENE SYNTHASE 1, PSY1) and anthocyanin regulatory gene (Ruby1, a MYB DOMAIN PROTEIN). On the other hand, it acted as a cofactor of HY5, enhancing the ability of HY5 to regulate target genes expression. Together, our results reveal the critical role of the transcriptional cascade consisting of BBX22 and HY5 in controlling internode elongation and pigment accumulation in citrus. Unraveling the crosstalk regulatory mechanism between internode elongation and fruit pigmentation provides key genes for breeding of novel types with both dwarf and health-beneficial fortification in citrus.

TRIPTYCHON-LIKE regulates aspects of both fruit flavor and color in citrus.

Deciphering the genetic basis of organoleptic traits is critical for improving the quality of fruits, which greatly shapes their appeal to consumers. Here, we characterize the citrus R3-MYB transcription factor TRIPTYCHON-LIKE (CitTRL), which is closely associated with the levels of citric acid, proanthocyanidins (PAs), and anthocyanins. Overexpression of CitTRL lowered acidity levels and PA contents in citrus calli as well as anthocyanin and PA contents in Arabidopsis leaves and seeds. CitTRL interacts with the two basic helix-loop-helix (bHLH) proteins CitbHLH1 and ANTHOCYANIN 1 (CitAN1) to regulate fruit quality. We show that CitTRL competes with the R2R3-MYB CitRuby1 for binding to CitbHLH1 or CitAN1, thereby repressing their activation of anthocyanin structural genes. CitTRL also competes with a second R2R3-MYB, CitPH4, for binding to CitAN1, thus altering the expression of the vacuolar proton-pump gene PH5 and Leucoanthocyanidin reductase, responsible for vacuolar acidification and proanthocyanidins biosynthesis, respectively. Moreover, CitPH4 activates CitTRL transcription, thus forming an activator-repressor loop to prevent the overaccumulation of citric acid and PAs. Overall, this study demonstrates that CitTRL acts as a repressor of the accumulation of citric acid, PAs, and anthocyanins by a cross-regulation mechanism. Our results provide an opportunity to simultaneously manipulate these key traits as a means to produce citrus fruits that are both visually and organoleptically appealing.

CsMYB3 and CsRuby1 form an 'Activator-and-Repressor' Loop for the Regulation of Anthocyanin Biosynthesis in Citrus.

Anthocyanins are preferentially accumulated in certain tissues of particular species of citrus. A R2R3-MYB transcription factor (named Ruby1) has been well documented as an activator of citrus anthocyanin biosynthesis. In this study, we characterized CsMYB3, a transcriptional repressor that regulates anthocyanin biosynthesis in citrus. CsMYB3 was expressed in anthocyanin-pigmented tissues, and the expression was closely associated with that of Ruby1, which is a key anthocyanin activator. Overexpression of CsMYB3 in Arabidopsis resulted in a decrease in anthocyanins under nitrogen stress. Overexpression of CsMYB3 in the background of CsRuby1-overexpressing strawberry and Arabidopsis reduced the anthocyanin accumulation level. Transient promoter activation assays revealed that CsMYB3 could repress the activation capacity of the complex formed by CsRuby1/CsbHLH1 for the anthocyanin biosynthetic genes. Moreover, CsMYB3 could be transcriptionally activated by CsRuby1 via promoter binding, thus forming an 'activator-and-repressor' loop to regulate anthocyanin biosynthesis in citrus. This study shows that CsMYB3 plays a repressor role in the regulation of anthocyanin biosynthesis and proposes an 'activator-and-repressor' loop model constituted by CsRuby1 and CsMYB3 in the regulation of anthocyanin biosynthesis in citrus.

Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults.

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H2O2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress.

Structure analysis and quality evaluation of plant-based meat analogs.

The growing world's population increases the demand of proteins. Meat products as the major source of high protein food are facing environmental impacts and animal welfare issues. Therefore, plant-based meat analogs are developed and gain a foothold in global markets. The structure design, sensory attributes and nutrient characteristics of meat analogs are crucial points to match the real meat. This review aimed to systematically introduce the structural analysis methods and evaluate meat analog products from quality-related attributes. First, various strategies of analyzing the fibrous structure of meat analogs were illustrated, including microscopic imaging and several optical techniques. Then, representative techniques such as NMR and AFM-IR for analyzing the distribution of moisture and lipid in meat analogs are introduced. In terms of quality, we elaborated on the texture and sensory evaluation methods and dialectically analyzed meat analogs' nutrition, which can provide a guidance for the advanced development of meat analogs.

The content and diversity of carotenoids associated with high-altitude adaptation in Tibetan peach fruit.

Carotenoids are important secondary metabolites that may participate in response to extreme environments. Fruit color changes were observed in peaches growing at altitude on the Tibetan Plateau. Here, we qualitatively and quantitatively analyzed 43 kinds of carotenoids in 96 Tibetan peach and 12 cultivated peach fruit samples. Comparative analysis revealed that 25 kinds of carotenoids accumulated at significantly different levels between Tibetan peaches and cultivated peaches. Based on a population structure analysis, the carotenoid levels of Tibetan peaches were divided into two groups, which are mainly affected by the environmental factors light and temperature. The correlation analysis implied that the levels of 9 carotenoids were significantly correlated with altitude. qRT-PCR results showed that PSY, CCD4 and BCH were significantly differently expressed between the low and high altitude Tibetan peaches. In summary, this study showed that the abundant variation in carotenoids was highly associated with high-altitude adaptations in Tibetan peach fruit.

SUMO1-regulated DBC1 promotes p53-dependent stress-induced apoptosis of lens epithelial cells.

Deleted in breast cancer 1 (DBC1) was initially identified from a homozygously deleted region in human chromosome 8p21. It has been well established that DBC1 plays a dual role during cancer development. Depending on the physiological context, it can promote or inhibit tumorigenesis. Whether it plays a role in lens pathogenesis remains elusive. In the present study, we demonstrated that DBC1 is highly expressed in lens epithelial cells from different vertebrates and in retina pigment epithelial cells as well. Moreover, DBC1 is SUMOylated through SUMO1 conjugation at K591 residue in human and mouse lens epithelial cells. The SUMOylated DBC1 is localized in the nucleus and plays an essential role in promoting stress-induced apoptosis. Silence of DBC1 attenuates oxidative stress-induced apoptosis. In contrast, overexpression of DBC1 enhances oxidative stress-induced apoptosis, and this process depends on p53. Mechanistically, DBC1 interacts with p53 to regulate its phosphorylation status at multiple sites and the SUMOylation of DBC1 enhances its interaction with p53. Together, our results identify that DBC1 is an important regulator mediating stress-induced apoptosis in lens, and thus participates in control of lens cataractogenesis.

Variation burst during dedifferentiation and increased CHH-type DNA methylation after 30 years of in vitro culture of sweet orange.

Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and induced via somatic embryogenesis. We compared four sweet orange callus lines after 30 years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitro culture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

Genome-wide identification of ovate family in Citrus and functional characterization of CitOFP19.

Fruit shape is an important trait for fruit appearance and commercial value. Diversity of fruit-shape has been utilized in the breeding of pummelo (Citrus maxima), a basic species in Citrus. However, little is known about genetic basis of fruit shape in citrus. In this study, we identified 16 OVATE family protein (OFP) genes in the pummelo genome. Phylogenetically, they were classified into three subfamilies, which was consistent with the classification of their Arabidopsis orthologs. Synteny analysis suggested that segment and tandem duplications were responsible for their expansion in pummelo. Expression pattern analysis of Citrus OFPs (CitOFPs) showed that CitOFP19 had significantly higher expression level in the ovaries of round pummelo than in those of pear-shaped pummelo. Heterologous overexpression of CitOFP19 in tomato resulted in pear-shaped ovary and fruit shape. Taken together, this study characterized OVATE gene family in Citrus genome and assessed the function of CitOFP19.

Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration.

Atrophic ("dry") form of age-related macular degeneration (AMD) is a leading cause of vision loss characterized by macular retinal pigment epithelium (RPE) and the ensuing photoreceptor degeneration. cGAS-STING signaling is a key cytosolic DNA sensor system in innate immunity and have recently been shown promotes RPE degeneration. However, expression regulation and therapeutic potential of cGAS and STING are not explored in retina under dry AMD pathogenic conditions. Our analysis shows upregulated STING RNA and increased chromatin accessibility around cGAS and STING promoters in macular retinas from dry AMD patients. cGAS-STING activation was detected in oxidative stress-induced mouse retina degeneration, accompanied with cytosolic leakage of damaged DNA in photoreceptors. Pharmaceutical or genetic approaches indicates STING promotes retina inflammation and degeneration upon oxidative damage. Drug screening reveals that BRD4 inhibitor JQ1 reduces cGAS-STING activation, inflammation and photoreceptor degeneration in the injured retina. BRD4 inhibition epigenetically suppresses STING transcription, and promotes autophagy-dependent cytosolic DNA clearance. Together, our results show that activation of cGAS-STING in retina may present pivotal innate immunity response in GA pathogenesis, whereas inhibition of cGAS-STING signaling by JQ1 could serve as a potential therapeutic strategy.

MYPT1/PP1-Mediated EZH2 Dephosphorylation at S21 Promotes Epithelial-Mesenchymal Transition in Fibrosis through Control of Multiple Families of Genes.

The methyltransferase EZH2 plays an important role in regulating chromatin conformation and gene transcription. Phosphorylation of EZH2 at S21 by AKT kinase suppresses its function. However, protein phosphatases responsible for the dephosphorylation of EZH2-S21 remain elusive. Here, it is demonstrated that EZH2 is highly expressed in the ocular lens, and AKT-EZH2 axis is important in TGFß-induced epithelial-mesenchymal transition (EMT). More importantly, it is identified that MYPT1/PP1 dephosphorylates EZH2-S21 and thus modulates its functions. MYPT1 knockout accelerates EMT, but expression of the EZH2-S21A mutant suppresses EMT through control of multiple families of genes. Furthermore, the phosphorylation status and gene expression modulation of EZH2 are implicated in control of anterior subcapsular cataracts (ASC) in human and mouse eyes. Together, the results identify the specific phosphatase for EZH2-S21 and reveal EZH2 dephosphorylation control of several families of genes implicated in lens EMT and ASC pathogenesis. These results provide important novel information in EZH2 function and regulation.

Generation of a homozygous CRISPR/Cas9-mediated knockout H9 hESC subline for the MERTK locus.

MERTK mutations are associate with rod-cone dystrophies. To enable investigations into the mechanism of this disease, we generated a cell line resource of H9 human embryonic stem cells harboring large fragment deletion mutation in a homozygous state in exon 19 of the MERTK gene. This subline expressed pluripotent stem cell markers, presented a normal karyotype, and preserved the ability to differentiate into endodermal, mesodermal, and ectodermal lineages.

A plant CitPITP1 protein-coding exon sequence serves as a promoter in bacteria.

Genetic manipulation of plant genes in prokaryotes has been widely used in molecular biology, but the function of a DNA sequence is far from being fully known. Here, we discovered that a plant protein-coding gene containing the CRAL_TRIO domain serves as a promoter in bacteria. We firstly characterized CitPITP1 from Citrus, which contains the CRAL_TRIO domain, and identified a 64-bp sequence (key64) that is critical for prokaryotic promoter activity. In vitro experiments indicated that the bacterial RNA polymerase subunit RpoD specifically binds to key64. We then expanded our research to fungi, plant and animal species to identify key64-like sequences. Five such prokaryotic promoters were isolated from Amborella, Rice, Arabidopsis and Citrus. Two conserved motifs were identified, and mutation analysis indicated that the nucleotides at positions 7, 29 and 30 are crucial for key64-like transcription activity. We detected full-length recombinant CitPITP1 from E. coli, and visualized a CitPITP1-GFP fusion protein in plant cells, supporting the idea that CitPITP1 encodes a protein. However, although exon 4 of CitPITP1 contained key64, it did not demonstrate promoter activity in plants. Our study describes a new basal promoter, provides evidence for neofunction of gene elements across different kingdoms, and provides new knowledge for the modular design of promoters.

Aging-dependent loss of GAP junction proteins Cx46 and Cx50 in the fiber cells of human and mouse lenses accounts for the diminished coupling conductance.

The homeostasis of the ocular lens is maintained by a microcirculation system propagated through gap junction channels. It is well established that the intercellular communications of the lens become deteriorative during aging. However, the molecular basis for this change in human lenses has not been well defined. Here, we present evidence to show that over 90% of Cx46 and Cx50 are lost in the fiber cells of normal human lenses aged 50 and above. From transparent to cataractous lenses, while Cx43 was upregulated, both Cx46 and Cx50 were significantly down-regulated in the lens epithelia. During aging of mouse lenses, Cx43 remained unchanged, but both Cx46 and Cx50 were significantly downregulated. Under oxidative stress treatment, mouse lenses develop in vitro cataractogenesis. Associated with this process, Cx43 was significantly upregulated, in contrast, Cx46 and Cx50 were sharply downregulated. Together, our results for the first time reveal that downregulation in Cx46 and Cx50 levels appears to be the major reason for the diminished coupling conductance, and the aging-dependent loss of Cx46 and Cx50 promotes senile cataractogenesis.

PP-1ß and PP-2Aα modulate cAMP response element-binding protein (CREB) functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53 signaling axis.

The function of the transcription factor, cAMP response element-binding protein (CREB), is activated through S133 phosphorylation by PKA and others. Regarding its inactivation, it is not well defined. cAMP response element-binding protein plays an essential role in promoting cell proliferation, neuronal survival and the synaptic plasticity associated with long-term memory. Our recent studies have shown that CREB is an important player in mediating stress response. Here, we have demonstrated that CREB regulates aging process through suppression of αB-crystallin and activation of the p300-p53-Bak/Bax signaling axis. First, we determined that two specific protein phosphatases, PP-1ß and PP-2Aα, can inactivate CREB through S133 dephosphorylation. Subsequently, we demonstrated that cells expressing the S133A-CREB, a mutant mimicking constant dephosphorylation at S133, suppress CREB functions in aging control and stress response. Mechanistically, S133A-CREB not only significantly suppresses CREB control of αB-crystallin gene, but also represses CREB-mediated activation of p53 acetylation and downstream Bak/Bax genes. cAMP response element-binding protein suppression of αB-crystallin and its activation of p53 acetylation are major molecular events observed in human cataractous lenses of different age groups. Together, our results demonstrate that PP-1ß and PP-2Aα modulate CREB functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53-Bax/Bak signaling axis, which regulates human cataractogenesis in the aging lens.

The E3 Ligase PIAS1 Regulates p53 Sumoylation to Control Stress-Induced Apoptosis of Lens Epithelial Cells Through the Proapoptotic Regulator Bax.

Protein sumoylation is one of the most important post-translational modifications regulating many biological processes (Flotho A & Melchior F. 2013. Ann Rev. Biochem. 82:357-85). Our previous studies have shown that sumoylation plays a fundamental role in regulating lens differentiation (Yan et al., 2010. PNAS, 107(49):21034-9.; Gong et al., 2014. PNAS. 111(15):5574-9). Whether sumoylation is implicated in lens pathogenesis remains elusive. Here, we present evidence to show that the protein inhibitor of activated STAT-1 (PIAS1), a E3 ligase for sumoylation, is implicated in regulating stress-induced lens pathogenesis. During oxidative stress-induced cataractogenesis, expression of PIAS1 is significantly altered at both mRNA and protein levels. Upregulation and overexpression of exogenous PIAS1 significantly enhances stress-induced apoptosis. In contrast, silence of PIAS1 with CRISPR/Cas9 technology attenuates stress-induced apoptosis. Mechanistically, different from other cells, PIAS1 has little effect to activate JNK but upregulates Bax, a major proapoptotic regulator. Moreover, Bax upregulation is derived from the enhanced transcription activity of the upstream transcription factor, p53. As revealed previously in other cells by different laboratories, our data also demonstrate that PIAS1 promotes SUMO1 conjugation of p53 at K386 residue in lens epithelial cells and thus enhances p53 transcription activity to promote Bax upregulation. Silence of Bax expression largely abrogates PIAS1-mediated enhancement of stress-induced apoptosis. Thus, our results demonstrated that PIAS1 promotes oxidative stress-induced apoptosis through positive control of p53, which specifically upregulates expression of the downstream proapoptotic regulator Bax. As a result, PIAS1-promoted apoptosis induced by oxidative stress is implicated in lens pathogenesis.

The transcription factor CREB acts as an important regulator mediating oxidative stress-induced apoptosis by suppressing αB-crystallin expression.

The general transcription factor, CREB has been shown to play an essential role in promoting cell proliferation, neuronal survival and synaptic plasticity in the nervous system. However, its function in stress response remains to be elusive. In the present study, we demonstrated that CREB plays a major role in mediating stress response. In both rat lens organ culture and mouse lens epithelial cells (MLECs), CREB promotes oxidative stress-induced apoptosis. To confirm that CREB is a major player mediating the above stress response, we established stable lines of MLECs stably expressing CREB and found that they are also very sensitive to oxidative stress-induced apoptosis. To define the underlying mechanism, RNAseq analysis was conducted. It was found that CREB significantly suppressed expression of the αB-crystallin gene to sensitize CREB-expressing cells undergoing oxidative stress-induced apoptosis. CREB knockdown via CRISPR/CAS9 technology led to upregulation of αB-crystallin and enhanced resistance against oxidative stress-induced apoptosis. Moreover, overexpression of exogenous human αB-crystallin can restore the resistance against oxidative stress-induced apoptosis. Finally, we provided first evidence that CREB directly regulates αB-crystallin gene. Together, our results demonstrate that CREB is an important transcription factor mediating stress response, and it promotes oxidative stress-induced apoptosis by suppressing αB-crystallin expression.

Molecular signature for senile and complicated cataracts derived from analysis of sumoylation enzymes and their substrates in human cataract lenses.

Sumoylation is one of the key regulatory mechanisms in eukaryotes. Our previous studies reveal that sumoylation plays indispensable roles during lens differentiation (Yan et al. 2010. Proc Natl Acad Sci USA. 107:21034-21039; Gong et al. 2014. Proc Natl Acad Sci USA. 111:5574-5579). Whether sumoylation is implicated in cataractogenesis, a disease largely derived from aging, remains elusive. In the present study, we have examined the changing patterns of the sumoylation ligases and de-sumoylation enzymes (SENPs) and their substrates including Pax6 and other proteins in cataractous lenses of different age groups from 50 to 90 years old. It is found that compared with normal lenses, sumoylation ligases 1 and 3, de-sumoylation enzymes SENP3/7/8, and p46 Pax6 are clearly increased. In contrast, Ubc9 is significantly decreased. Among different cataract patients from 50s to 70s, male patients express more sumoylation enzymes and p46 Pax6. Ubc9 and SENP6 display age-dependent increase. The p46 Pax6 displays age-dependent decrease in normal lens, remains relatively stable in senile cataracts but becomes di-sumoylated in complicated cataracts. In contrast, sumoylation of p32 Pax6 is observed in senile cataracts and increases its stability. Treatment of rat lenses with oxidative stress increases Pax6 expression without sumoylation but promotes apoptosis. Thus, our results show that the changing patterns in Ubc9, SENP6, and Pax6 levels can act as molecular markers for senile cataract and the di-sumoylated p46 Pax6 for complicated cataract. Together, our results reveal the presence of molecular signature for both senile and complicated cataracts. Moreover, our study indicates that sumoylation is implicated in control of aging and cataractogenesis.