RESUMO
Aldo-keto reductases (AKRs) are a superfamily of promiscuous enzymes that have been chiseled by evolution to act as catalysts for numerous regulatory pathways in humans. However, they have not lost their promiscuity in the process, essentially making them a double-edged sword. The superfamily is involved in multiple metabolic pathways and are linked to chronic diseases such as cataracts, diabetes, and various cancers. Unlike other detoxifying enzymes such as cytochrome P450s (CYP450s), short-chain dehydrogenases (SDRs), and medium-chain dehydrogenases (MDRs), that participate in essential pathways, AKRs are more widely distributed and have members with interchangeable functions. Moreover, their promiscuity is ubiquitous across all species and participates in the resistance of pathogenic microbes. Moreover, the introduction of synthetic substrates, such as synthetic molecules and processed foods, results in unwanted "toxification" due to enzyme promiscuity, leading to chronic diseases.
Assuntos
Aldo-Ceto Redutases , Catarata , Neoplasias , Humanos , Aldo-Ceto Redutases/metabolismo , Aldo-Ceto Redutases/genética , Catarata/enzimologia , Catarata/genética , Catarata/metabolismo , Doença Crônica , Neoplasias/enzimologia , Neoplasias/genéticaRESUMO
The purpose of this study was to investigate whether and how topical nerve growth factor (NGF) attenuates streptozotocin (STZ)-induced diabetic cataracts in vivo. Rats were randomly divided into three groups, including the normal control rat group, STZ-induced diabetic cataract rat group (DM group), and STZ-induced diabetic cataract rat group treated with 200 µg/mL recombinant rat ß-NGF (DM + NGF group). Cataract formation was evaluated by portable slit lamp biomicroscopy following pupil dilation at 8 weeks. The expression levels of NGF, aldose reductase (AR), and Na+/K+-ATPase in the lens epithelial cells (LECs) of the three groups were measured in the presence or absence of topical NGF. TUNEL-positive LECs were quantified to determine if hyperglycemia caused LEC apoptosis. At 8 weeks, the mean cataract score in the control group was significantly lower than that in DM and DM + NGF groups, and the score in the DM + NGF group was significantly lower than that in the DM group. At the equatorial zone and anterior central zone of lens, NGF and Na+/K+-ATPase expression levels were significantly decreased in the DM group; however, they were partially restored in the DM + NGF group. At the equatorial zone and anterior central zone of lens, AR expression and TUNEL-positive apoptotic LECs were significantly increased in the DM group compared with the control group, however, they were significantly decreased in the DM + NGF group. In conclusion, topical NGF could delay the progression of diabetic cataracts by attenuating polyol pathway activation and increasing Na+/K+-ATPase protein levels.
Assuntos
Catarata/prevenção & controle , Diabetes Mellitus Experimental/prevenção & controle , Fator de Crescimento Neural/uso terapêutico , ATPase Trocadora de Sódio-Potássio/metabolismo , Administração Oftálmica , Animais , Apoptose , Catarata/induzido quimicamente , Catarata/enzimologia , Catarata/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Hiperglicemia/metabolismo , Marcação In Situ das Extremidades Cortadas , Masculino , Soluções Oftálmicas , Polímeros , Ratos , Ratos Sprague-Dawley , Estreptozocina/toxicidade , Regulação para CimaRESUMO
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.
Assuntos
Catarata/metabolismo , Catarata/patologia , Sumoilação/fisiologia , Envelhecimento/fisiologia , Apoptose , Catarata/enzimologia , Diferenciação Celular/fisiologia , Feminino , Humanos , Cristalino/enzimologia , Cristalino/metabolismo , Cristalino/patologia , Ligases/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Lycium barbarum polysaccharide (LBP) extracted from the Lycium barbarum L. has been widely used to improve diabetes and its relative complications. However, the mechanisms have not fully understood. A recent study has demonstrated that LBP upregulates suituin 1 (SIRT1). OBJECTIVE: This study was to define the role of Sirt1 and its downstream signaling pathways in diabetic cataract using in vitro and in vivo models. MATERIALS AND METHODS: Human lens epithelial cell line SRA01/04 cells were cultured under high glucose (HG) medium with treatment of LBP or vehicle. Cell viability, apoptosis, protein and/or mRNA levels of Sirt1, BAX, Bcl-2, active-caspase-3, FOXO1, p27 and acetylated p53 were measured. SIRT1 upregulated- and knocked-down cells were generated and tested in high glucose culture. Diabetes mellitus was induced in rats by streptozotocin injection. Body weight, blood glucose levels, lens transparency and retinal function were assessed and SIRT1, as well as the aforementioned biomarkers were measured using Western blotting and qPCR in the animal lens samples. RESULTS: The results showed that HG decreased cell viability and LBP prevented the decrease. The reduced viability in HG cultured SRA01/04 cells was associated with increased levels of BAX, active caspase 3, FOXO1, p27, and p53 and decreased levels of SIRT1 and Bcl-2. Further experiments using sirt1 gene modulated cells showed that upregulation of Sirt1 improved viability, increase cell division as reflected by an increased proportion of S phase in the cell cycle, reduced the number of apoptotic cell death and suppressed p53 acetylation and caspase 3 activation. Opposite results were observed in SIRT1 knock-down cells. Treating diabetic animals with LBP reduced body weight loss and blood glucose content in diabetic animals. Similarly, LBP hindered the development of cataract in lenses and improved retinal function. The beneficial effect of LBP on diabetic cataract was associated with the supression of p53, caspase 3, FOXO1, BAX, p27 and elevation of SIRT1 and Bcl-2, which were consistent with the in vitro findings. CONCLUSION: Our findings showed that diabetes caused cataract is associated with suppression of SIRT1 and Bcl-2 and activation of other cell death related genes. LBP prevented diabetic cataract in animals by upregulating Sirt1 and Bcl-2 and suppressing cell death related genes.
Assuntos
Catarata/prevenção & controle , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Células Epiteliais/efeitos dos fármacos , Cristalino/efeitos dos fármacos , Lycium , Sirtuína 1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Catarata/enzimologia , Catarata/etiologia , Catarata/patologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Medicamentos de Ervas Chinesas/isolamento & purificação , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Cristalino/enzimologia , Cristalino/patologia , Lycium/química , Masculino , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/genéticaRESUMO
Purpose: To identify the genetic defect in a four-generation Chinese family that causes autosomal dominant congenital posterior subcapsular cataracts, and to understand how this EPHA2 kinase domain mutation affects EPHA2 activity. Methods: Variants in 54 cataract-associated genes were screened by targeted next generation sequencing (NGS) and then validated by Sanger sequencing. EPHA2 wild-type cDNA was synthesized in vitro, and EPHA2 p.G668D mutant was constructed by PCR site-directed mutagenesis. Western blotting and fluorescence microscopy were used to analyze the expression level of protein and its subcellular localization, respectively. A wound-healing assay was performed to analyze changes to cell migration. Results: A novel heterozygous missense mutation was identified in the kinase domain of the EPHA2 gene (c.2003G>A, p.G668D). This is the third congenital cataract mutation being reported in this domain. Functional study revealed that the kinase domain mutation (p.G668D) decreased EphA2 protein level (P = 0.036) via a proteasome-dependent pathway, altered its subcellular localization of the EphA2 from cell-cell contacts to a diffuse perimembranous distribution, and changed the distribution of ß-catenin as well. The expression of mutant EphA2 significantly promoted the migration of human lens epithelial cells (P = 0.002). Conclusions: Our study presented the evidence for a novel EPHA2 kinase domain mutation that causes congenital posterior subcapsular cataracts. The first functional study on an EPHA2 kinase domain mutation that causes a congenital cataract revealed that the G668D mutation destabilized the receptor, changed its subcellular localization, and altered the activation of EphA2 with its ligand ephrin. The mutant EphA2 resulted in a reduced inhibition of cell migration. As a consequence, the c.G668D mutation promoted cell migration and caused the formation of cataracts.
Assuntos
Catarata/congênito , Catarata/genética , Efrina-A2/metabolismo , Mutação de Sentido Incorreto , Receptor EphA2/genética , Povo Asiático/genética , Western Blotting , Catarata/enzimologia , Catarata/patologia , Movimento Celular/fisiologia , Células Cultivadas , China/epidemiologia , Análise Mutacional de DNA , Células Epiteliais/metabolismo , Feminino , Vetores Genéticos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Lentivirus/genética , Masculino , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Linhagem , Plasmídeos , Reação em Cadeia da Polimerase , Receptor EphA2/metabolismo , beta Catenina/metabolismoRESUMO
Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.
Assuntos
Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/genética , Catarata/enzimologia , Catarata/genética , Hipogonadismo/enzimologia , Hipogonadismo/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Pirofosfatases/deficiência , Animais , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Homozigoto , Humanos , Inosina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/enzimologia , Mutação , Linhagem , Pirofosfatases/genética , RNA/genética , RNA/metabolismo , Sequenciamento do ExomaRESUMO
The endocannabinoid (eCB) system is an important part of both the human central nervous system (CNS) and peripheral tissues. It is involved in the regulation of various physiological and neuronal processes and has been associated with various diseases. The eCB system is a complex network composed of receptor molecules, their cannabinoid ligands, and enzymes regulating the synthesis, release, uptake, and degradation of the signalling molecules. Although the eCB system and the molecular processes of eCB signalling have been studied extensively over the past decades, the involved molecules and underlying signalling mechanisms have not been described in full detail. An example pose the two poorly characterised eCB-degrading enzymes α/ß-hydrolase domain protein six (ABHD6) and ABHD12, which have been shown to hydrolyse 2-arachidonoyl glycerol-the main eCB in the CNS. We review the current knowledge about the eCB system and the role of ABHD6 and ABHD12 within this important signalling system and associated diseases. Homology modelling and multiple sequence alignments highlight the structural features of the studied enzymes and their similarities, as well as the structural basis of disease-related ABHD12 mutations. However, homologies within the ABHD family are very low, and even the closest homologues have widely varying substrate preferences. Detailed experimental analyses at the molecular level will be necessary to understand these important enzymes in full detail.
Assuntos
Endocanabinoides/metabolismo , Metabolismo dos Lipídeos/fisiologia , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Doenças Neurodegenerativas/enzimologia , Animais , Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Ataxia/enzimologia , Ataxia/etiologia , Catarata/enzimologia , Catarata/etiologia , Biologia Computacional , Endocanabinoides/química , Glicerídeos/química , Glicerídeos/metabolismo , Humanos , Monoacilglicerol Lipases/genética , Mutação , Polineuropatias/enzimologia , Polineuropatias/etiologia , Alcamidas Poli-Insaturadas/química , Alcamidas Poli-Insaturadas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Retinose Pigmentar/enzimologia , Retinose Pigmentar/etiologia , Transdução de Sinais/fisiologiaRESUMO
Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase that has been shown to be involved in the pathogenesis of several blinding diseases such as uveitis, diabetic retinopathy (DR) and cataract. However, possible mechanisms linking the action of AR to these diseases are not well understood. As DR and cataract are among the leading causes of blindness in the world, there is an urgent need to explore therapeutic strategies to prevent or delay their onset. Studies with AR inhibitors and gene-targeted mice have demonstrated that the action of AR is also linked to cancer onset and progression. In this review we examine possible mechanisms that relate AR to molecular signaling cascades and thus explain why AR inhibition is an effective strategy against colon cancer as well as diseases of the eye such as uveitis, cataract, and retinopathy.
Assuntos
Aldeído Redutase/metabolismo , Catarata/enzimologia , Retinopatia Diabética/enzimologia , Inflamação/enzimologia , Uveíte/enzimologia , Aldeído Redutase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/uso terapêutico , Camundongos , Transdução de SinaisRESUMO
Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC) is a rare genetic human neurological disorder caused by null mutations to the Abhd12 gene, which encodes the integral membrane serine hydrolase enzyme ABHD12. Although the role that ABHD12 plays in PHARC is understood, the thorough biochemical characterization of ABHD12 is lacking. Here, we report the facile synthesis of mono-1-(fatty)acyl-glycerol lipids of varying chain lengths and unsaturation and use this lipid substrate library to biochemically characterize recombinant mammalian ABHD12. The substrate profiling study for ABHD12 suggested that this enzyme requires glycosylation for optimal activity and that it has a strong preference for very-long-chain lipid substrates. We further validated this substrate profile against brain membrane lysates generated from WT and ABHD12 knockout mice. Finally, using cellular organelle fractionation and immunofluorescence assays, we show that mammalian ABHD12 is enriched on the endoplasmic reticulum membrane, where most of the very-long-chain fatty acids are biosynthesized in cells. Taken together, our findings provide a biochemical explanation for why very-long-chain lipids (such as lysophosphatidylserine lipids) accumulate in the brains of ABHD12 knockout mice, which is a murine model of PHARC.
Assuntos
Ataxia/enzimologia , Catarata/enzimologia , Lipídeos/química , Monoacilglicerol Lipases/química , Polineuropatias/enzimologia , Retinose Pigmentar/enzimologia , Animais , Ataxia/genética , Ataxia/metabolismo , Encéfalo/enzimologia , Encéfalo/metabolismo , Catarata/genética , Catarata/metabolismo , Humanos , Cinética , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Polineuropatias/genética , Polineuropatias/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Especificidade por SubstratoRESUMO
Reactive oxygen species and lipid peroxidation are important factors that contribute to the development of age-related cataract. The study included 130 patients with age-related cataract, 69 of whom were diagnosed with hypertension (HT), 20 with hypertension and type 2 diabetes mellitus (DM), and 41 had no accompanying condition. The following parameters were measured in the serum of the examinees: products of lipid peroxidation malondialdehyde (MDA) and lipofuscin-like fluorophores (LLF), activity of prooxidative enzymes xanthine oxidase (XO) and myeloperoxidase (MPO), antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), the concentration of thiol groups, and the ferric reducing activity of plasma. The activity of prooxidative enzymes XO and MPO was higher in the plasma of patients with HT (XO=9.0±1.2 U/L; MPO=77.3±8.4 U/L) and with HT and DM (XO=11.9±0.9 U/L; MPO=89.5±5.0 U/L) compared to patients with age-related cataract (XO=6.2±0.9 U/L; MPO=52.4±6.3 U/L; P<0.01). Our research has shown that patients with age-related cataract and hypertension were exposed to increased oxidative damage of biomolecules, based on the increased plasma LLF and MDA content and decreased levels of thiol groups. Oxidative changes of biomolecules in these patients were associated with increased activity of the XO, MPO, and GPx enzymes and a lower extracellular SOD activity and total ferric reductive ability of plasma.
Assuntos
Catarata/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Hipertensão/enzimologia , Xantina Oxidase/metabolismo , Idoso , Biomarcadores/sangue , Catarata/complicações , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Hipertensão/complicações , Masculino , Xantina Oxidase/sangueRESUMO
PURPOSE: Protein sumoylation is a well established regulatory mechanism that regulates chromatin structure and dynamics, cell proliferation and differentiation, stress response and cell apoptosis. In the vertebrate eye, we and others have shown that sumoylation plays an indispensable role in regulating eye development. During stress induction and aging process, the ocular tissues gradually loss their normality and develop major ocular diseases such as cataract and aging-related macular degeneration. We have recently demonstrated that sumoylation actively regulates differentiation of lens cells, whether this process is implicated in lens pathogenesis remains to be investigated. In this study, we have demonstrated that transparent mouse lenses treated with glucose oxidase and UVA irradiation undergo in vitro cataract formation, and associated with this process, the expression patterns of the 3 sumoylation enzymes have been found significantly altered. METHODS: Four-week-old C57BL/6J mice were used in our experiment. Lenses were carefully excised from eyes and cultured in M199 medium (Sigma 3769) for at least 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 30 mU/mL glucose oxidase (GO, MP Biomedicals, 1673) to induce cataract formation. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: we have obtained the following results: 1) Both GO treatment and UVA irradiation can induce cataract formation in the in vitro cultured mouse lenses; 2) With GO treatment, the mRNAs and proteins for the 5 sumoylation enzymes were all significantly downregulated; 3) With UVA irradiation, the changes in the expression patterns of the mRNAs and proteins for the SAE1, UBA2 , UBC9 and PIAS1 were opposite, while the mRNAs were upregulated either significantly (for SAE1, UBA2 and UBC9) or slightly (PIAS1), the proteins for all 4 sumoylation enzymes were downregulated; For RanBP2, the UVA induced changes in both mRNA and protein are consist with the GO treatment. CONCLUSION: Under GO and UVA irradiation conditions, the expression levels of both mRNA and protein for the three major sumoylation enzymes were significantly changed. Our results suggest that altered expression patterns of the sumoylation enzymes are associated with oxidative stressinduced cataractogenesis.
Assuntos
Catarata , Regulação Enzimológica da Expressão Gênica/imunologia , Glucose Oxidase , Cristalino , Sumoilação , Enzimas Ativadoras de Ubiquitina , Raios Ultravioleta/efeitos adversos , Animais , Catarata/enzimologia , Catarata/imunologia , Catarata/patologia , Glucose Oxidase/imunologia , Glucose Oxidase/metabolismo , Cristalino/enzimologia , Cristalino/imunologia , Cristalino/patologia , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/imunologia , Sumoilação/imunologia , Sumoilação/efeitos da radiação , Enzimas Ativadoras de Ubiquitina/biossíntese , Enzimas Ativadoras de Ubiquitina/imunologiaRESUMO
Reactive oxygen species and lipid peroxidation are important factors that contribute to the development of age-related cataract. The study included 130 patients with age-related cataract, 69 of whom were diagnosed with hypertension (HT), 20 with hypertension and type 2 diabetes mellitus (DM), and 41 had no accompanying condition. The following parameters were measured in the serum of the examinees: products of lipid peroxidation malondialdehyde (MDA) and lipofuscin-like fluorophores (LLF), activity of prooxidative enzymes xanthine oxidase (XO) and myeloperoxidase (MPO), antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), the concentration of thiol groups, and the ferric reducing activity of plasma. The activity of prooxidative enzymes XO and MPO was higher in the plasma of patients with HT (XO=9.0±1.2 U/L; MPO=77.3±8.4 U/L) and with HT and DM (XO=11.9±0.9 U/L; MPO=89.5±5.0 U/L) compared to patients with age-related cataract (XO=6.2±0.9 U/L; MPO=52.4±6.3 U/L; P<0.01). Our research has shown that patients with age-related cataract and hypertension were exposed to increased oxidative damage of biomolecules, based on the increased plasma LLF and MDA content and decreased levels of thiol groups. Oxidative changes of biomolecules in these patients were associated with increased activity of the XO, MPO, and GPx enzymes and a lower extracellular SOD activity and total ferric reductive ability of plasma.
Assuntos
Humanos , Masculino , Idoso , Xantina Oxidase/metabolismo , Catarata/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Hipertensão/enzimologia , Xantina Oxidase/sangue , Catarata/complicações , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/complicações , Hipertensão/complicaçõesRESUMO
BACKGROUND & OBJECTIVES: The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus. METHODS: Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction. RESULTS: IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P<0.05) but not in diabetic retina, and there was an increase in PON- HCTLase activity (P<0.05) only in diabetic retina. Bioinformatic studies and in vitro experiments indicated that advanced glycation end products (AGE) such as carboxymethyl -lysine might decrease the PON- AREase activity of the PON. INTERPRETATION & CONCLUSIONS: Distribution of PON enzyme and its activity in ocular tissues is reported here. The study revealed maximal PON activity in lens and retina, which are prone to higher oxidative stress. Differential activities of PON were observed in the lens and retinal tissues from cataractous and diabetic patients, respectively.
Assuntos
Arildialquilfosfatase/genética , Catarata/genética , Retinopatia Diabética/genética , Adulto , Idoso , Antioxidantes/metabolismo , Catarata/enzimologia , Catarata/patologia , Retinopatia Diabética/enzimologia , Retinopatia Diabética/patologia , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Produtos Finais de Glicação Avançada/genética , Humanos , Cristalino/enzimologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Retina/enzimologia , Retina/patologiaRESUMO
Mutations in mitochondrial acylglycerol kinase (AGK) cause Sengers syndrome, which is characterized by cataracts, hypertrophic cardiomyopathy, and skeletal myopathy. AGK generates phosphatidic acid and lysophosphatidic acid, bioactive phospholipids involved in lipid signaling and the regulation of tumor progression. However, the molecular mechanisms of the mitochondrial pathology remain enigmatic. Determining its mitochondrial interactome, we have identified AGK as a constituent of the TIM22 complex in the mitochondrial inner membrane. AGK assembles with TIMM22 and TIMM29 and supports the import of a subset of multi-spanning membrane proteins. The function of AGK as a subunit of the TIM22 complex does not depend on its kinase activity. However, enzymatically active AGK is required to maintain mitochondrial cristae morphogenesis and the apoptotic resistance of cells. The dual function of AGK as lipid kinase and constituent of the TIM22 complex reveals that disturbances in both phospholipid metabolism and mitochondrial protein biogenesis contribute to the pathogenesis of Sengers syndrome.
Assuntos
Cardiomiopatias/enzimologia , Catarata/enzimologia , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Translocador 1 do Nucleotídeo Adenina/metabolismo , Antiporters/metabolismo , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/patologia , Catarata/genética , Catarata/patologia , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos , Mutação , Fenótipo , Fosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico , Fatores de Tempo , TransfecçãoRESUMO
Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.
Assuntos
Cardiomiopatias/enzimologia , Catarata/enzimologia , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cardiomiopatias/genética , Catarata/genética , Ciclo do Ácido Cítrico , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Estabilidade Proteica , Transporte Proteico , TransfecçãoRESUMO
PURPOSE: To study the relationships between matrix metalloproteinases (MMP)-2, MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3 aqueous humor levels in patients with high myopia or cataract. MATERIALS AND METHODS: MMPs and TIMPs protein levels in 65 aqueous humor samples collected from patients with high myopia or cataract during cataract or clear lens extraction surgery were measured with the Luminex xMAP Technology. The relationship between MMPs and TIMPs levels was analyzed with Spearman's correlation test. RESULTS: MMP-2 levels, but not MMP-3 levels, were increased in the aqueous humor from high-myopia patients. Levels of TIMP-1, -2, and -3 were positively and very significantly correlated with the MMP-2 levels (TIMP-1: r=0.626, p < 0.001; TIMP-2: r = 0.545, p < 0.001; TIMP-3: r = 0.439, p < 0.001). TIMP-2 and-3 levels did not significantly correlate with MMP-3 levels (TIMP-2: r = 0.175, p > 0.05; TIMP-3: r = 0.127, p > 0.05) and TIMP-1 levels only marginally correlated with MMP-3 levels (r = 0.278, 0.01< P < 0.05). CONCLUSIONS: Compared to the present findings with the relationship of MMPs and TIMPs in other fields of medicine, our results are consistent with the homeostasis hypothesis that the increase of TIMPs serves as a compensation reaction to inhibit the excessive degradation caused by the increase of MMPs and limits the development of myopia.
Assuntos
Humor Aquoso/enzimologia , Catarata/enzimologia , Metaloproteinases da Matriz/metabolismo , Miopia Degenerativa/enzimologia , Inibidores Teciduais de Metaloproteinases/metabolismo , Adulto , Comprimento Axial do Olho/patologia , Feminino , Humanos , Imunoensaio/métodos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Estatística como Assunto , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
PURPOSE: To discern and confirm genetic biomarkers that help identify populations at high risk for age-related cataract (ARC). METHODS: A literature search was performed in the PubMed, Web of Science and China National Knowledge Internet databases for genetic association studies published before June 26, 2016 regarding ARC susceptibility. All genetic polymorphisms reported were systematically reviewed, followed by extraction of candidate genes/loci with sufficient genotype data in ≥3 studies for the meta-analysis. A random/fixed-effects model was used to calculate the pooled odds ratios and 95% confidence intervals to evaluate the associations considering multiple genetic models. Sensitivity analysis was also performed. RESULTS: A total of 144 polymorphisms in 36 genes were reported in the 61 previous genetic association studies. Thereby, three polymorphisms of two genes (8-oxoguanine DNA glycosylase-1 [OGG1]; methylenetetrahydrofolate reductase NADPH [MTHFR]) in eight studies were included in the meta-analysis. Regarding the OGG1-rs1052133, the GG (OR = 1.925; 95%CI, 1.181-3.136; p = 0.009) and CG (OR = 1.384; 95%CI, 1.171-1.636; p<0.001) genotypes indicated higher risk of ARC. For the MTHFR gene, the CC+TT genotype of rs1801133 might be protective (OR, 0.838; 95%CI, 0.710-0.989; p = 0.036), whereas the AA+CC genotype of rs1801131 indicated increased risk for the mixed subtype (OR = 1.517; 95%CI, 1.113-2.067; p = 0.008). CONCLUSIONS: Polymorphisms of OGG1 and MTHFR genes are associated with ARC susceptibility and may help identify populations at high risk for ARC.
Assuntos
Catarata/enzimologia , Catarata/genética , DNA Glicosilases/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Predisposição Genética para Doença/genética , HumanosRESUMO
The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFCD). In contrast to the disassembly and reassembly of the cell nucleus during mitosis, LFCD is a unidirectional process that culminates in destruction of the fiber cell nucleus. Whereas p27Kip1, the cyclin-dependent kinase inhibitor, is upregulated during formation of LFC in the outermost cortex, in the inner cortex, in the nascent organelle free zone, p27Kip1 is degraded, markedly activating cyclin-dependent kinase 1 (Cdk1). This process results in phosphorylation of nuclear Lamins, dissociation of the nuclear membrane, and entry of lysosomes that liberate DNaseIIß (DLAD) to cleave chromatin. Multiple cellular pathways, including the ubiquitin proteasome system and the unfolded protein response, converge on post-translational regulation of p27Kip1. Mutations that impair these pathways are associated with congenital cataracts and loss of LFCD. These findings highlight new regulatory nodes in the lens and suggest that we are close to understanding this fascinating terminal differentiation process. Such knowledge may offer a new means to confront proliferative diseases including cancer.
Assuntos
Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Cristalino/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Catarata/congênito , Catarata/enzimologia , Catarata/patologia , Humanos , Laminas/metabolismo , Cristalino/citologia , Cristalino/enzimologia , Mitose , FosforilaçãoRESUMO
OBJECTIVE: To investigate whether a single nucleotide polymorphism (SNP) rs1136410 in the poly (ADP-ribose) polymerase-1 (PARP-1) gene was associated with PARP activities, 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels, and the risk of age-related cataract (ARC) in a Chinese Han population. METHODS: In this two-stage case-control study with a total of 1010 ARC patients and 1045 controls, SNP rs1136410 was genotyped by high-resolution melting analyses (HRM). PARP activities and 8-OHdG levels in peripheral blood mononuclear cells (PBMCs) were determined by ELISA kits. RESULTS: In discovery, replication, and their merged sets, the variant genotypes (AG+GG) of SNP rs1136410 were significantly associated with an increased risk of ARC under a dominant model (Adjusted odds ratio (OR)=1.42, Padj=0.001 for the merged set). This association was further identified in subtype analyses for cortical ARC (Adjusted OR=1.69, Padj<0.001). In subgroup analyses, we identified a significant interaction between SNP rs1136410 and smoking habit in increasing ARC risk (Pinter=0.019). Moreover, ARC patients had lower activities of PARP and higher levels of 8-OHdG than controls. There were significant correlations of SNP rs1136410 with decreased PARP activities and increased 8-OHdG levels in controls and patients with cortical ARC. CONCLUSIONS: This study suggests that SNP rs1136410 may confer susceptibility to ARC by affecting PARP activities and oxidative DNA damage.