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.     

Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.Age-related macular degeneration (AMD)¹ is one of the most common pathologies in the retina, consisting in a chronic degenerative disorder that constitutes the leading cause of blindness in the elderly, probably affecting 14 million people in the United States. AMD is a multifactorial disease in nature in which age is the predominant risk factor, although there are also environmental factors involved. In this regard, smoking is thought to be a major environmental risk factor as supported by extensive epidemiological evidence (1–5). AMD develops in two different stages: early AMD (also referred to as dry AMD) and the late stage of AMD known as wet AMD by virtue of the extensive neovascularization taking place in the retina choroid. Although there is a fair understanding of the mechanisms involved in wet AMD, little is known about dry AMD and its transition into the most severe stage of this disorder, i.e. wet AMD (6).Early AMD targets the retinal pigment epithelium (RPE) and the Bruch membrane (BrM) in the retina. The RPE constitutes a cell monolayer that is crucial to maintain a normal photoreceptor function. In fact, RPE participates in the cycling of the visual molecules, provides nutrients to rods and cones, and is responsible for withdrawing waste debris from the outer segments of photoreceptors (7). The early stage of AMD is characterized by initial deregulation of the normal extracellular matrix (ECM) turnover leading to thickening of the BrM, sub-RPE deposit accumulation, and drusen formation (8). As mentioned earlier, cumulative evidence suggests that smoking may constitute a major risk factor for early AMD. In fact, we and others have provided evidence that hydroquinone (HQ), a major component of cigarette smoke, has the ability to deregulate the ECM (9–12). Aside from cigarette smoke, HQ is a compound of environmental relevance because of its broad presence in plastics, foodstuff, and air pollution (13, 14).Mild injuries inflected to the retina elicit a cellular response in the RPE consisting in pinching off small areas of the plasma membrane, which renders small microvesicles called blebs (15). The reason(s) behind membrane blebbing remains unknown, although it has been postulated to be an attempt to discard damaged cellular constituents by the RPE cell (8). Under prolonged injury, blebs may accumulate between the RPE and the basal lamina underneath this cell monolayer. Based on this concept, a plausible role for blebs in the pathogenesis of dry AMD has been suggested as a likely contributor to build-up of the sub-RPE deposits, which are characteristic of the early stages of this disorder (8). To date, however, RPE bleb composition and potential functions remain largely unexplored.However, membrane bleb or microvesicle production stimulated by a variety of stress has been extensively described in many different cell types (16–23). To gain a better understanding of the functional relevance of blebs in general and the pathogenic mechanism(s) involved in early AMD in particular, we sought to investigate the identity of proteins carried by human RPE blebs. Previously microvesicles from lymphocytes have been subjected to analysis leading to the identification of a number of proteins (24). In our study, we show the proteomics characterization of stress-induced blebs in RPE cells from human retina. We report identification of several proteins, some of them potentially involved in matrix metalloproteinase (MMP) activation, membrane lipid raft formation, and immunogenic processes. Interestingly RPE blebs were found to carry basigin (including highly glycosylated species) and MMP-14, which are key proteins regulating the ECM turnover and remodeling. A previous proteomics study also has revealed the presence of basigin in the blebs from malignant lymphocytes (24). In the present study, we intended to gain some insight into the functional characterization of blebs to unravel some of the biological consequences of cell membrane blebbing in disease.     

Quantitative Proteomics: Comparison of the Macular Bruch Membrane/Choroid Complex from Age-related Macular Degeneration and Normal Eyes

A quantitative proteomics analysis of the macular Bruch membrane/choroid complex was pursued for insights into the molecular mechanisms of age-related macular degeneration (AMD). Protein in trephine samples from the macular region of 10 early/mid-stage dry AMD, six advanced dry AMD, eight wet AMD, and 25 normal control post-mortem eyes was analyzed by LC MS/MS iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 901 proteins was quantified, including 556 proteins from ≥3 AMD samples. Most proteins differed little in amount between AMD and control samples and therefore reflect the proteome of normal macular tissues of average age 81. A total of 56 proteins were found to be elevated and 43 were found to be reduced in AMD tissues relative to controls. Analysis by category of disease progression revealed up to 16 proteins elevated or decreased in each category. About 60% of the elevated proteins are involved in immune response and host defense, including many complement proteins and damage-associated molecular pattern proteins such as α-defensins 1–3, protein S100s, crystallins, histones, and galectin-3. Four retinoid processing proteins were elevated only in early/mid-stage AMD, supporting a role for retinoids in AMD initiation. Proteins uniquely decreased in early/mid-stage AMD implicate hematologic malfunctions and weakened extracellular matrix integrity and cellular interactions. Galectin-3, a receptor for advanced glycation end products, was the most significantly elevated protein in advanced dry AMD, supporting a role for advanced glycation end products in dry AMD progression. The results endorse inflammatory processes in both early and advanced AMD pathology, implicate different pathways of progression to advanced dry and wet AMD, and provide a new database for hypothesis-driven and discovery-based studies of AMD.Age-related macular degeneration (AMD)¹ is a leading cause of blindness worldwide (1, 2) and is approaching epidemic proportions in the United States (3). AMD is a progressive disease involving multiple genetic and environmental factors. Deposition of debris (termed “drusen”) along Bruch membrane in the macula is the first evidence of early AMD. Advanced AMD occurs in two forms, geographic atrophy and choroidal neovascularization (CNV). Geographic atrophy (advanced dry AMD) develops slowly and results in blindness when focal areas of the retinal pigment epithelium (RPE) degenerate in the macula. CNV (wet AMD) is characterized by the growth of new blood vessels from the choroid through Bruch membrane and the RPE. When these vessels hemorrhage, a blood clot accumulates between the RPE and the macular photoreceptors causing immediate central vision loss. CNV accounts for over 80% of debilitating visual loss in AMD, yet only 10–15% of AMD cases progress to wet AMD.There is growing evidence that AMD is in part an age-related inflammatory disease involving complement dysregulation, including AMD susceptibility genes encoding complement factors and the presence of complement proteins in drusen (1, 2, 4). An assortment of potential inducers of AMD have been proposed; however, the causes of the disease remain poorly defined. For example, many carrying AMD risk genotypes may never develop the disease (5), and only a fraction of those diagnosed with early AMD progress to advanced disease (6). We have proposed that oxidative protein modifications are among the catalysts of AMD (7), and a host of elevated oxidative modifications have been reported in AMD Bruch membrane, drusen, retina, RPE, and plasma (7–14). Two of these modifications, namely carboxymethyllysine and carboxyethylpyrrole (CEP), stimulate neovascularization in vivo (15, 16), and immunization with CEP-adducted protein has been shown to induce a dry AMD-like phenotype in mice (17). Yet many other factors have been reported to contribute to this complex disease, including for example cigarette smoking (18), cumulative light exposure (19), lipofuscin/retinoid toxicity (20), advanced glycation end products (8–10), and microbial infection (21).Toward a better understanding of the molecular pathways contributing to AMD pathology, we pursued quantitative proteomics analysis of the macular region of the Bruch membrane/choroid complex. Bruch membrane (see Fig. 1) is a stratified extracellular matrix (ECM) comprising a central elastin zone flanked by inner and outer collagenous layers and the basement membranes of the RPE and choriocapillaris (22). It serves as a semipermeable support for the RPE, which forms an integral part of the blood-retinal barrier and provides many vital functions for vision, including the rod retinoid visual cycle and phagocytosis of spent photoreceptors with export of degradation products to the blood. The Bruch membrane molecular sieve is thought to help regulate the diffusion of nutrients and waste products between the RPE and the bloodstream as well as to restrict cell migration (23). Age-related changes at this critical interface, including thickening and decreased permeability, have long been thought to disrupt normal retinal physiology and contribute to AMD (22, 23). We used LC MS/MS iTRAQ technology to quantify proteins from a relatively large number of age- and gender-matched AMD and normal macular tissues and to correlate proteomic changes with AMD progression. The results endorse inflammatory processes in AMD pathology, reveal molecular details previously unassociated with AMD, and provide a quantitative proteomics database from the critically important macular interface.Open in a separate windowFig. 1.Macular Bruch membrane tissue samples. Bruch membrane is a permeable extracellular matrix separating the RPE from the blood-bearing choroid as illustrated in a cross-section of the human eye (A) and a cross-section through the macular region (B). Photographs are shown of isolated Bruch membrane tissue before (C) and after (D) removal of 4-mm trephined tissue buttons for proteomics analysis (F, fovea) scale bar = 2.4 mm. A and B are reproduced with copyright permission from the Cleveland Clinic. Illustration by David Schumick. All rights reserved.     

Pigment Epithelium-derived Factor Maintains Retinal Pigment Epithelium Function by Inhibiting Vascular Endothelial Growth Factor-R2 Signaling through ��-Secretase

Wet age-related macular degeneration (AMD) attacks the integrity of the retinal pigment epithelium (RPE) barrier system. The pathogenic process was hypothesized to be mediated by vascular endothelial growth factor (VEGF) and antagonized by pigment epithelium-derived factor (PEDF). To dissect these functional interactions, monolayer cultures of RPE cells were established, and changes in transepithelial resistance were evaluated after administration of PEDF, placenta growth factor (VEGF-R1 agonist), and VEGF-E (VEGF-R2 agonist). A recently described mechanism of VEGF inhibition in endothelia required the release of VEGF-R1 intracellular domain by γ-secretase. To evaluate this pathway in the RPE, cells were pretreated with inhibitors DAPT or {"type":"entrez-nucleotide","attrs":{"text":"LY411575","term_id":"1257853995","term_text":"LY411575"}}LY411575. Processing of VEGF receptors was assessed by Western blot analysis. Administration of VEGF-E rapidly increased RPE permeability, and PEDF inhibited the VEGF-E response dose-dependently. Both γ-secretase antagonists prevented the inhibitory effects of PEDF. The co-administration of PEDF and VEGF-E depleted the amount of VEGF-R2 in the membrane and increased the amount of VEGF-R2 ectodomain in the media. Therefore, the inhibitory effect of PEDF appears to be mediated via the processing of VEGF-R2 by γ-secretase. γ-Secretase generates the amyloid-β (Aβ) peptide of Alzheimer disease from its precursor (amyloid precursor protein). This peptide is also a component of drusen in dry AMD. The results support the hypothesis that misregulation of γ-secretase may not only lead to Aβ deposits in dry AMD but can also be damaging to RPE function by blocking the protective effects of PEDF to prevent VEGF from driving the dry to wet AMD transition.Age-related macular degeneration (AMD)² is often diagnosed by the appearance of subretinal fluid. This fluid causes a local detachment of the retina in the macular area resulting in decreased visual acuity in the center of the visual field (1). The resulting macular edema can lead to complete vision loss (2). Although the excessive fluid mainly comes from capillaries in the inner retina, the removal of subretinal fluid is dependent on the RPE. The maintenance of RPE barrier function is essential for the efficient removal of the fluid (3), and the disruption of the RPE barrier can eventually lead to choroidal neovascularization.Recent clinical studies have shown that intravitreally administered anti-VEGF compounds are effective therapies for choroidal neovascularization (4–6). Originally, VEGF was described as an endothelial angiogenic and vasopermeability factor. The leakage through the vessels of the inner retina increases in response to VEGF (7, 8). However, the release of VEGF also affects RPE function (9–11). We have recently shown that RPE barrier integrity is modulated by VEGF through apically oriented VEGF-R2 receptors (12). Thus, there is a growing body of evidence that intraocular VEGF can increase the permeability of both the inner and outer blood-retina barriers, contributing to the accumulation of subretinal fluid and macular edema.Pigment epithelium-derived factor was initially identified as a neurotrophic agent secreted by fetal human RPE cells (13). Subsequent experiments have recognized that PEDF is an endogenous antagonist of VEGF (14). In the eye, studies have provided evidence that endothelial quiescence and barrier function is achieved through a balance of VEGF and PEDF (15). The PEDF secretion pattern from the RPE cells is predominantly apical, and the interphotoreceptor matrix around the RPE microvilli is a major reservoir of PEDF (16, 17). Therefore, we hypothesize that PEDF can antagonize the breakdown of RPE function induced by the apical actions of VEGF.Several schemes have been proposed for the anti-VEGF activity of PEDF. A PEDF receptor has been identified, which has phospholipase A₂ activity (18). PEDF binding proteins without clear receptor activity have also been found (19). In endothelial cells, PEDF has also been shown to compete with VEGF for binding at the VEGF-R2 receptor (20). PEDF was found to regulate VEGF expression (20, 21) and decrease VEGF receptor phosphorylation (14). A recent study in endothelia has elucidated a novel inhibitory mechanism of VEGF signaling via the PEDF-induced intramembrane proteolysis of VEGF-R1 by γ-secretase (22). The goal of our study is to determine if PEDF acts as an anti-permeability agent in the RPE and to begin to understand the cellular mechanism involved in this response.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Question Processing and Clustering in INDOC: A Biomedical Question Answering System

The exponential growth in the volume of publications in the biomedical domain has made it impossible for an individual to keep pace with the advances. Even though evidence-based medicine has gained wide acceptance, the physicians are unable to access the relevant information in the required time, leaving most of the questions unanswered. This accentuates the need for fast and accurate biomedical question answering systems. In this paper we introduce INDOC—a biomedical question answering system based on novel ideas of indexing and extracting the answer to the questions posed. INDOC displays the results in clusters to help the user arrive the most relevant set of documents quickly. Evaluation was done against the standard OHSUMED test collection. Our system achieves high accuracy and minimizes user effort.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]     

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.Insights into many cellular processes require detailed information about interactions between the participating proteins. However, the analysis of such interactions can be challenging because of the often-diverse physicochemical properties and the abundances of the constituent proteins, as well as the sometimes wide range of affinities and complex dynamics of the interactions. One of the key challenges has been acquiring information concerning transient, low affinity interactions in highly complex cellular milieux (3, 4).Methods that allow elucidation of such information include co-localization microscopy (5), fluorescence protein Förster resonance energy transfer (4), immunoelectron microscopy (5), yeast two-hybrid (6), and affinity capture (7, 8). Among these, affinity capture (AC)¹ has the unique potential to detect all specific in vivo interactions simultaneously, including those that interact both directly and indirectly. In recent times, the efficacy of such affinity isolation experiments has been greatly enhanced through the use of sensitive modern mass spectrometric protein identification techniques (9). Nevertheless, AC suffers from several shortcomings. These include the problem of 1) distinguishing specific from nonspecific interactors (10, 11); 2) preserving and isolating all unique interactions including those that are weak and/or transient, as well as those that exchange rapidly (10, 12, 13); and 3) differentiating proximal from more distant interactions (14).We describe here an approach to address these issues, which makes use of chemical stabilization of protein assemblies in the complex cellular milieu prior to AC. Chemical stabilization is an emerging technique for stabilizing and elucidating protein associations both in vitro (15–20) and in vivo (3, 12, 14, 21–29), with mass spectrometric (MS) readout of the AC proteins and their connectivities. Such chemical stabilization methods are indeed well-established and are often used in electron microscopy for preserving complexes and subcellular structures both in the cellular milieu (3) and in purified complexes (30, 31), wherein the most reliable, stable, and established stabilization reagents is glutaraldehyde. Recently, glutaraldehyde has been applied in the “GraFix” protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30, 31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.In this work, we present a robust pipeline for determining specific protein-protein interactions and proximities from cellular milieux. The first steps of the pipeline involve the well-established techniques of flash freezing the cells of interest in liquid nitrogen and cryomilling, which have been known for over a decade (33, 34) to preserve the cellular environment, as well as having shown outstanding performance when used in analysis of macromolecular interactions in yeast (35–39), bacterial (40, 41), trypanosome (42), mouse (43), and human (44–47) systems. The resulting frozen powder, composed of intact micron chunks of cells that have great surface area and outstanding solvent accessibility, is well suited for rapid low temperature chemical stabilization using glutaraldehyde. We selected glutaraldehyde for our procedure based on the fact that it is a very reactive stabilizing reagent, even at lower temperatures, and because it has already been shown to stabilize enzymes in their functional state (48–50). We employed highly efficient, rapid, single stage affinity capture (36, 51) for isolation and bottom-up MS for analysis of the macromolecular assemblies of interest (52–54). For convenience, we have termed this approach Stabilized Affinity-Capture Mass Spectrometry (SAC-MS).