Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.     

The role of PIWIL4 and piRNAs in the development of choroidal neovascularization

Wet age-related macular degeneration (wAMD) is the leading cause of blindness among the elderly in industrialized nations. Anti-vascular epidermal growth factor (VEGF) therapy via intravitreal injection is the most effective clinical treatment for wAMD due to high concentrations of VEGF that promote choroidal neovascularization. While PIWI proteins participate in various biological processes, their function in AMD remains unclear. In this study, we discovered that PIWIL4 expression is elevated in a laser-induced choroidal neovascularization (CNV) model and that it regulates angiogenesis in vitro and in vivo. Differentially expressed piwi-interacting RNAs (piRNAs) were identified in a CNV model and were shown to potentially regulate angiogenesis via bioinformatics analysis. PIWIL4 knockdown inhibits VEGF secretion and VEGFR2 phosphorylation. Overall, PIWIL4 may serve as a novel target to block pathological choroidal neovascularization, and the study of the PIWI-piRNAs pathway in wAMD highlights its broad function in somatic cells.     

The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.     

Blockade of receptor for advanced glycation endproducts: a new target for therapeutic intervention in diabetic complications and inflammatory disorders

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states.     

Effects of advanced glycation end products-inductor glyoxal and hydrogen peroxide as oxidative stress factors on rat retinal organ cultures and neuroprotection by UK-14,304

Retinal ganglion cell degeneration is supposed to be mediated by reactive oxygen species (ROS) and advanced glycation end products (AGEs). The alpha2-adrenergic agonist, 5-bromo- N -(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (brimonidine; UK-14,304), is said to exert a neuroprotective effect. To investigate these mechanisms in detail, we exposed rat whole mounts to glyoxal or H₂O₂ and treated them with either UK-14,304 alone or additionally with the phosphatidylinositide 3 kinase (PI3) kinase inhibitor, 2-(4-Morpholinyl)-8-phenyl-4 H -1-benzopyran-4-one (Ly 294002). The accumulation of Nε-[carboxymethyl] lysine (CML) was assessed immunohistochemically and changes in intracellular pH (pHi), mitochondrial transmembrane potential (MTMP) and ROS production in cell bodies of multipolar ganglion cell layer were studied by intravital fluorescence microscopy and confocal laser scanning microscopy. Ultrastructural changes in mitochondria of multipolar ganglion cell layer cell bodies were determined by transmission electron microscopy. We found that glyoxal and H₂O₂ increased accumulation of CML-modified proteins and ROS production and decreased pHi and MTMP in cell bodies of multipolar ganglion cell layer. UK-14,304 could prevent production of ROS, accumulation of CML-modified proteins, ameliorate acidification, preserve MTMP and attenuate ultrastructural damages of ganglion cell mitochondria. Ly 294002 reversed the UK-14,304-mediated attenuation of CML and ROS production. We conclude that the protective effects of UK-14,304 seem partly to be mediated by PI3 kinase-dependent pathways.     

Plasma Protein Pentosidine and Carboxymethyllysine, Biomarkers for Age-related Macular Degeneration

Age-related macular degeneration (AMD) causes severe vision loss in the elderly; early identification of AMD risk could help slow or prevent disease progression. Toward the discovery of AMD biomarkers, we quantified plasma protein N^ε-carboxymethyllysine (CML) and pentosidine from 58 AMD and 32 control donors. CML and pentosidine are advanced glycation end products that are abundant in Bruch membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaris. We measured CML and pentosidine by LC-MS/MS and LC-fluorometry, respectively, and found higher mean levels of CML (∼54%) and pentosidine (∼64%) in AMD (p < 0.0001) relative to normal controls. Plasma protein fructosyl-lysine, a marker of early glycation, was found by amino acid analysis to be in equal amounts in control and non-diabetic AMD donors, supporting an association between AMD and increased levels of CML and pentosidine independent of other diseases like diabetes. Carboxyethylpyrrole (CEP), an oxidative modification from docosahexaenoate-containing lipids and also abundant in AMD Bruch membrane, was elevated ∼86% in the AMD cohort, but autoantibody titers to CEP, CML, and pentosidine were not significantly increased. Compellingly higher mean levels of CML and pentosidine were present in AMD plasma protein over a broad age range. Receiver operating curves indicate that CML, CEP adducts, and pentosidine alone discriminated between AMD and control subjects with 78, 79, and 88% accuracy, respectively, whereas CML in combination with pentosidine provided ∼89% accuracy, and CEP plus pentosidine provided ∼92% accuracy. Pentosidine levels appeared slightly altered in AMD patients with hypertension and cardiovascular disease, indicating further studies are warranted. Overall this study supports the potential utility of plasma protein CML and pentosidine as biomarkers for assessing AMD risk and susceptibility, particularly in combination with CEP adducts and with concurrent analyses of fructosyl-lysine to detect confounding factors.Age-related macular degeneration (AMD)¹ is a progressive, multifactorial disease and a major cause of severe vision loss in the elderly (1). Deposition of debris (drusen) in the macular region of Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE), is an early, hallmark risk factor of AMD. The disease can progress to advanced dry AMD (geographic atrophy), which is characterized by regional degeneration of photoreceptor and RPE cells, or to advanced wet AMD (choroidal neovascularization (CNV)), which is characterized by abnormal blood vessels growing from the choriocapillaris through Bruch membrane beneath the retina. CNV accounts for over 80% of debilitating vision loss in AMD; however, only 10–15% of AMD cases progress to CNV.There is growing consensus that AMD is an age-related inflammatory disease involving dysregulation of the complement system; however, triggers of the inflammatory response have yet to be well defined. Oxidative stress appears to be involved as smoking significantly increases the risk of AMD (2), antioxidant vitamins can selectively slow AMD progression (3), and a host of oxidative protein and DNA modifications have been detected at elevated levels in AMD Bruch membrane, drusen, retina, RPE, and plasma (4–11). Oxidative protein modifications like carboxyethylpyrrole (CEP) and N^ε-carboxymethyllysine (CML), both elevated in AMD Bruch membrane, stimulate neovascularization in vivo (12, 13), suggesting possible roles in CNV. Other studies have shown that mice immunized with CEP protein modifications develop an AMD-like phenotype (14). Accordingly oxidative modifications may be catalysts or triggers of AMD pathology (6).AMD has long been hypothesized to be a systemic disease (15) based in part on the presence of retinal drusen in patients with membranoproliferative glomerulonephritis type II (16) and systemic complement activation in AMD (17). Support for this hypothesis also comes from mounting evidence that advanced glycation end products (AGEs) may play a role in AMD (4, 5, 7, 18, 19). AGEs are a heterogeneous group of mostly oxidative modifications resulting from the Maillard nonenzymatic glycation reaction that have been associated with age-related diseases and diabetic complications (20, 21). In 1998, CML was the first AGE to be found in AMD Bruch membrane and drusen (4). Other AGEs have since been detected in AMD ocular tissues (5, 7, 18) and in Bruch membrane, drusen, RPE, and choroidal extracellular matrix from healthy eyes (6, 22). CML, a nonfluorescent AGE, and pentosidine, a fluorescent cross-linking AGE, increase with age in Bruch membrane (18, 23). Receptors for AGEs (RAGE and AGE-R1) appear elevated on RPE and photoreceptor cells in early and advanced dry AMD (7) especially in RPE overlying drusen-like deposits on Bruch membrane (19). AGE-R3, also known as galectin-3, is elevated in AMD Bruch membrane (24).Although AMD susceptibility genes now account for over 50% of AMD cases (25), many individuals with AMD risk genotypes may never develop advanced disease with severe vision loss. Nevertheless the prevalence of advanced AMD is increasing (26). Toward the discovery of better methods to detect those at risk for advanced AMD, we quantified CML and pentosidine in plasma proteins from AMD and control patients and compared their discriminatory accuracy with plasma CEP biomarkers. CEP biomarkers have been shown to enhance the AMD predictive accuracy of genomic AMD biomarkers (11). This report shows CML and pentosidine to be elevated in AMD plasma proteins and demonstrates their potential biomarker utility in assessing AMD risk and susceptibility especially in combination with CEP biomarkers.     

Alu DNA polymorphism in ACE gene is protective for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. We report an association between an Alu polymorphism in the angiotensin-converting enzyme (ACE) gene with the dry/atrophic form of AMD. Using the polymerase chain reaction (PCR) on genomic DNA isolated from patients with AMD (n=173), and an age-matched control population (n=189), we amplified a region polymorphic for an Alu element insertion in the ACE gene. The Alu(+/+) genotype occurred 4.5 times more frequently in the control population than the dry/atrophic AMD patient population, (p=0.004). The predominance of the Alu(+/+) genotype within the unaffected control group represents a protective insertion with respect to the human ocular disease, dry/atrophic AMD. This is the first demonstration of an Alu element insertion exerting protective effects against a known human disease.     

Increased level of glycoxidation product N(epsilon)-(carboxymethyl)lysine in rat serum and urine proteins with aging: link with glycoxidative damage accumulation in kidney

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43+/-0.14 pmol CML/micrograms protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50+/-0.14 pmol CML/micrograms protein vs 2.27+/-0.26 pmol CML/micrograms protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50+/-0.14 pmol CML/micrograms protein at 3 months and markedly increased from 10 months (2.99+/-0.24 pmol CML/micrograms protein) to 27 months (3.76+/-0.25 pmol CML/micrograms protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowman's capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85+/-28.95 pmol CML/microgrms OHPro at 3 months and significantly increased from 10 months (187.61+/-21.99 pmol CML/micrograms OHPro) to 27 months (334.55+/-62.21 pmol CML/micrograms OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.     

Carboxyethylpyrrole protein adducts and autoantibodies,biomarkers for age-related macular degeneration

Age-related macular degeneration (AMD) is a slow, progressive disease with both genetic and environmental risk factors. Free radical-induced oxidation of docosahexaenoate (DHA)-containing lipids generates omega-(2-carboxyethyl)pyrrole (CEP) protein adducts that are more abundant in ocular tissues from AMD than normal human donors. To understand better the role of oxidative damage in AMD, we have synthesized CEP-modified proteins, produced anti-CEP antibodies, and initiated analysis of CEP immunoreactivity and autoantibodies in human plasma. A highly selective rabbit polyclonal anti-CEP antibody was raised that binds CEP 1000 times more strongly than carboxypropylpyrrole, a close structural analogue. The CEP adduct uniquely indicates oxidative modification from DHA derivatives because CEP protein modifications cannot arise from any other common polyunsaturated fatty acid. Immunocytochemistry localized CEP to photoreceptor rod outer segments and retinal pigment epithelium in mouse retina and demonstrated more intense CEP immunoreactivity in photoreceptors from a human AMD donor compared with healthy human retina. The mean level of anti-CEP immunoreactivity in AMD human plasma (n = 19 donors) was 1.5-fold higher (p = 0.004) than in age-matched controls (n = 19 donors). Sera from AMD patients demonstrated mean titers of anti-CEP autoantibody 2.3-fold higher than controls (p = 0.02). Of individuals (n = 13) exhibiting both antigen and autoantibody levels above the mean for non-AMD controls, 92% had AMD. These results suggest that together CEP immunoreactivity and autoantibody titer may have diagnostic utility in predicting AMD susceptibility.     

Plasma Peptide Biomarker Discovery for Amyotrophic Lateral Sclerosis by MALDI –TOF Mass Spectrometry Profiling

The diagnostic of Amyotrophic lateral sclerosis (ALS) remains based on clinical and neurophysiological observations. The actual delay between the onset of the symptoms and diagnosis is about 1 year, preventing early inclusion of patients into clinical trials and early care of the disease. Therefore, finding biomarkers with high sensitivity and specificity remains urgent. In our study, we looked for peptide biomarkers in plasma samples using reverse phase magnetic beads (C18 and C8) and MALDI-TOF mass spectrometry analysis. From a set of ALS patients (n=30) and healthy age-matched controls (n=30), C18- or C8-SVM-based models for ALS diagnostic were constructed on the base of the minimum of the most discriminant peaks. These two SVM-based models end up in excellent separations between the 2 groups of patients (recognition capability overall classes > 97%) and classify blinded samples (10 ALS and 10 healthy age-matched controls) with very high sensitivities and specificities (>90%). Some of these discriminant peaks have been identified by Mass Spectrometry (MS) analyses and correspond to (or are fragments of) major plasma proteins, partly linked to the blood coagulation.     

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

大鼠2/3肝切除72 h后再生肝脏质膜比较蛋白质组学研究

大鼠2/3肝切除模型为研究肝细胞增殖和生理性血管生成提供了一个很好的活体内模型.为了揭示肝再生过程中与肝细胞增殖终止相关及与血管生成启动相关的质膜蛋白质,本研究对大鼠肝2/3部分切除72 h后的肝脏质膜进行了研究：利用两步蔗糖密度梯度离心法对切除组和假手术组的肝脏质膜进行纯化;然后通过双向电泳和质谱技术对肝切除样品进行了比较分析并对几个关键蛋白程序性凋亡相关蛋白-6和丝蛋白-A进行了免疫印迹验证.相对于假手术对照组(Sham组),21种蛋白质在切除后72 h的肝脏中上调,15种蛋白质下调.所鉴定的差异表达蛋白参与了血管生成、细胞分裂增殖和凋亡、细胞分化调控、肝脏组织重新构建、代谢及应急反应.本研究为肝脏再生及其血管生成的研究提供了理论依据.     

Changes in rat liver mitochondria with aging. Lon protease-like reactivity and N(epsilon)-carboxymethyllysine accumulation in the matrix.

Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was measured and the accumulation of oxidation and glycoxidation (Nepsilon-carboxymethyllysine, CML) products was monitored using immunochemical assays. The function of isolated mitochondria was assessed by measuring respiratory chain activity. Mitochondria from aged (27 months) rats exhibited the same rate of oxygen consumption as those from adult (10 months) rats without any change in coupling efficiency. At the same time, the ATP-stimulated Lon protease activity, measured as fluorescent peptides released, markedly decreased from 10-month-old rats (1.15 +/- 0.15 FU x micro g protein-1 x h-1) to 27-month-old-rats (0.59 +/- 0.08 FU x micro g protein-1 x h-1). In parallel with this decrease in activity, oxidized proteins accumulated in the matrix upon aging while the CML-modified protein content assessed by ELISA significantly increased by 52% from 10 months (11.71 +/- 0.61 pmol CML x micro g protein-1) to 27 months (17.81 +/- 1.83 pmol CML x micro g protein-1). These results indicate that the accumulation of deleterious oxidized and carboxymethylated proteins in the matrix concomitant with loss of the Lon protease activity may affect the ability of aging mitochondria to respond to additional stress.     

Chorionic expression of heterogeneous products of the PAG (Pregnancy-Associated Glycoprotein) gene family secreted in vitro throughout embryonic and foetal development in the pig

Porcine PAG (pPAG) are placental products of a multigene family that is strongly expressed in the chorionic epithelium (trophoblast and trophectoderm). The objective of this study was to define a pattern of the pPAG proteins, secreted in vitro by chorionic explants harvested on 16-77 days of pregnancy. Trophoblastic and trophectodermal explants were collected from pregnant (PR) gilts (n = 27) and used for protein in vitro production (8-261 h). Endometrial explants of luteal-phase gilts (E10, n = 4) and pseudopregnant gilts (PsE, n = 2) were used as negative controls for protein immunoblotting. Proteins (PR, E10, PsE) were isolated mainly from incubation media, fractionated, dialysed and separated by SDS-PAGE. Heterogeneous Western blotting with various polyclonal anti-PAG sera raised against bovine or ovine antigens (anti-bPAG, or anti-oPAG) initially identified the pPAG proteins. Such blotting of fractionated chorionic proteins allowed for the isolation of porcine antigens that were employed as immunogens to raise several homologous antisera (anti-pPAG). Crude antisera were adsorbed on endometrial extracts or proteins of non-PR pigs, to remove non-relevant antibodies. The patterns of pPAG proteins secreted in vitro varied throughout pregnancy (35-72 kDa). During implantation, approximately 43 kDa (Day 16) or approximately 68.1 kDa (Days 17-25) pPAG proteins were detected. During placentation and as pregnancy advanced (Days 31-77), approximately 72.3 kDa pPAG proteins were observed. The secretions of parallel multiple smaller proteins (35.4-47.2 kDa), presumably, as forms of processed pPAG precursors, increased with the progress of gestation. In conclusion, the pPAG protein family plays a very important role during implantation, placenta formation and embryonic/foetal development in the pig.     

An altered pattern of circulating apolipoprotein E3 isoforms is implicated in preeclampsia

Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.     

Plasma protein glycation in Alzheimer's disease

Recent studies have suggested that formation of advanced glycation end-products (AGEs) in some brain proteins could be associated with Alzheimer's disease.These AGEs can be produced by various sugars (hexose, pentose, glyceraldehyde and oxidative products of vitamin C). In this study, we quantified plasma protein glycation specifically derived from glucose in patients with Alzheimer's disease with different grades of cognitive disorders.Two groups of Alzheimer patients were studied: a group with moderate Alzheimer's disease (n=6, 9相似文献   

Nuclear proteasome activation and degradation of carboxymethylated histones in human keratinocytes following glyoxal treatment

Nuclear DNA damage has been studied in detail, but much less is known concerning the occurrence and fate of nuclear protein damage. Glycoxidation, protein damage that results from a combination of protein glycation and oxidation, leads to the formation of protein-advanced glycation end products (AGE) of which N(epsilon)-carboxymethyllysine (CML) is a major AGE. We have used glyoxal, a product of environmental exposures that readily leads to the formation of CML, to study nuclear protein glycoxidation in HaCaT human keratinocytes. Glyoxal treatment that did not affect cell viability but inhibited cell proliferation in a dose-dependent manner that led to accumulation of CML-modified histones. Modified histones were slowly degraded but persisted for more than 3 days following treatment. Preincubation of cells with a proteasome inhibitor following glyoxal treatment led to an increase in CML-modified histones. While glyoxal treatment resulted in a slight decrease in total cellular proteasome activity, a dose dependent increase of up to 4-fold in nuclear proteasome activity was observed. The increase in nuclear proteasome activity was due to both increased nuclear proteasome protein content and increased activity, neither of which were affected by cyclohexamide. The increase also was unaffected by inhibitors of poly(ADP-ribose) polymerases, which have been previously implicated in nuclear proteasome activation by oxidizing agents. Accumulation of CML-modified histones over time may lead to epigenetic changes that contribute to various pathologies including aging and cancer, and upregulation of nuclear proteasome activity under conditions of glyoxidative stress may function to limit such damage.     

Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore? analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.     

Conventional antibody against Nepsilon-(carboxymethyl)lysine (CML) shows cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL): immunochemical quantification of CML with a specific antibody

Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.     

HTRA1, an age‐related macular degeneration protease,processes extracellular matrix proteins EFEMP1 and TSP1

High‐temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age‐related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high‐risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high‐risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP‐1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.