Biosynthetic Processing Research Articles

Functions and Mechanisms of Pro-Lysyl Oxidase Processing in Cancers and Eye Pathologies with a Focus on Diabetic Retinopathy.

Lysyl oxidases are multifunctional proteins derived from five lysyl oxidase paralogues (LOX) and lysyl oxidase-like 1 through lysyl oxidase-like 4 (LOXL1–LOXL4). All participate in the biosynthesis of and maturation of connective tissues by catalyzing the oxidative deamination of lysine residues in collagens and elastin, which ultimately results in the development of cross-links required to function. In addition, the five LOX genes have been linked to fibrosis and cancer when overexpressed, while tumor suppression by the propeptide derived from pro-LOX has been documented. Similarly, in diabetic retinopathy, LOX overexpression, activity, and elevated LOX propeptide have been documented. The proteolytic processing of pro-forms of the respective proteins is beginning to draw attention as the resultant peptides appear to exhibit their own biological activities. In this review we focus on the LOX paralogue, and what is known regarding its extracellular biosynthetic processing and the still incomplete knowledge regarding the activities and mechanisms of the released lysyl oxidase propeptide (LOX-PP). In addition, a summary of the roles of both LOX and LOX-PP in diabetic retinopathy, and brief mentions of the roles for LOX and closely related LOXL1 in glaucoma, and keratoconus, respectively, are included.

Control of membrane protein homeostasis by a chaperone-like glial cell adhesion molecule at multiple subcellular locations

The significance of crosstalks among constituents of plasma membrane protein clusters/complexes in cellular proteostasis and protein quality control (PQC) remains incompletely understood. Examining the glial (enriched) cell adhesion molecule (CAM), we demonstrate its chaperone-like role in the biosynthetic processing of the megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1)-heteromeric regulatory membrane protein complex, as well as the function of the GlialCAM/MLC1 signalling complex. We show that in the absence of GlialCAM, newly synthesized MLC1 molecules remain unfolded and are susceptible to polyubiquitination-dependent proteasomal degradation at the endoplasmic reticulum. At the plasma membrane, GlialCAM regulates the diffusional partitioning and endocytic dynamics of cluster members, including the ClC-2 chloride channel and MLC1. Impaired folding and/or expression of GlialCAM or MLC1 in the presence of diseases causing mutations, as well as plasma membrane tethering compromise the functional expression of the cluster, leading to compromised endo-lysosomal organellar identity. In addition, the enlarged endo-lysosomal compartments display accelerated acidification, ubiquitinated cargo-sorting and impaired endosomal recycling. Jointly, these observations indicate an essential and previously unrecognized role for CAM, where GliaCAM functions as a PQC factor for the MLC1 signalling complex biogenesis and possess a permissive role in the membrane dynamic and cargo sorting functions with implications in modulations of receptor signalling.

Classifying molecular phenotypes of G6PC variants for pathogenic properties and to guide therapeutic development.

Due to advances in sequencing technologies, identification of genetic variants is rapid. However, the functional consequences of most genomic variants remain unknown. Consequently, variants of uncertain significance (VUS) that appear in clinical DNA diagnostic reports lack sufficient data for interpretation. Algorithms exist to aid prediction of a variant's likelihood of pathogenicity, but these predictions usually lack empiric evidence. To examine the feasibility of generating functional evidence in vitro for a given variant's role in disease, a panel of 29 coding sequence variants in the G6PC gene was assessed. G6PC encodes glucose‐6 phosphatase enzyme, and reduction in its function causes the rare metabolic disease glycogen storage disease type 1a (GSD1a). Variants were heterologously expressed as fusion proteins in a hepatocyte‐derived cell line and examined for effects on steady‐state protein levels, biosynthetic processing, and intracellular distribution. The screen revealed variant effects on protein levels, N‐linked glycosylation status, and cellular distribution. Of the eight VUS tested, seven behaved similar to wild‐type protein while one VUS, p.Cys109Tyr, exhibited features consistent with pathogenicity for all molecular phenotypes assayed, including significantly reduced protein levels, alteration in protein glycosylation status, and abnormally diffuse protein localization pattern, and has recently been reported in a patient with GSD1a. Our results show that such a screen can add empiric evidence to existing databases to aid in diagnostics, and also provides further classification for molecular phenotypes that could be used in future therapeutic screening approaches for small molecule or gene editing strategies directed at specific variants.

Proteinase bone morphogenetic protein 1, but not tolloid-like 1, plays a dominant role in maintaining periodontal homeostasis.

Periodontitis is caused by multiple factors involving a bacterial challenge and a susceptible host, although there is no report on gene mutation directly linked to this common disease. Mutations in the proteinase bone morphogenetic protein 1 (BMP1) were identified in patients with osteogenesis imperfecta, who display some dentin defects and alveolar bone loss. We previously reported essential roles of BMP1 and tolloid-like 1 (TLL1), two closely related extracellular proteinases with overlapping functions, in mouse periodontium growth by simultaneous knockout (KO) of both genes, although the separate roles of BMP1 and TLL1 have remained unclear. Here, we have investigated whether and how BMP1 and TLL1 separately maintain periodontal homeostasis by comparing single Bmp1 KO and Tll1 KO with double KO (dKO) phenotypes. Floxed Bmp1 and/or Tll1 alleles were deleted in transgenic mice via ubiquitously expressed CreERT2 induced by tamoxifen treatment starting at 4-weeks of age (harvested at 18-weeks of age). Multiple approaches, including X-ray, micro-CT, calcein and alizarin red double-labeling, scanning electron microscopy, and histological and immunostaining assays, were used to analyze periodontal phenotypes and molecular mechanisms. Both Bmp1 KO and double KO mice exhibited severe periodontal defects, characterized by periodontal ligament (PDL) fiber loss and ectopic ossification in the expanded PDL area, and drastic reductions in alveolar bone and cementum volumes, whereas Tll1 KO mice displayed very mild phenotypes. Mechanistic studies revealed a sharp increase in the uncleaved precursor of type I collagen (procollagen I), leading to defective extracellular matrices. BMP1, but not TLL1, is essential for maintaining periodontal homeostasis. This occurs at least partly via biosynthetic processing of procollagen I, thereby maintaining appropriate levels of procollagen I and its activated products such as mature collagen I.

Role of bone morphogenetic protein 1/tolloid proteinase family in the development of teeth and bone

The bone morphogenetic protein (BMP) 1/tolloid (TLD) proteinase family is a group of important metalloproteinases, which play key roles in the growth and development of tissues and organs via regulating the biosynthetic processing of the extracellular matrix. Clinical reports have revealed that mutations in the genes encoding BMP1/TLD proteinases lead to dentinogenesis imperfecta type Ⅰ, accompanied with osteogenesis imperfecta. Therefore, this proteinase family is essential for the development of hard tissues. In this study, we review the research progress in the function and mechanism of the BMP1/TLD proteinase family in the development of teeth and bone.

Step-Economic Synthesis of Biomimetic β-Ketopolyene Thioesters and Demonstration of Their Usefulness in Enzymatic Biosynthesis Studies.

Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic β-ketopolyene thioesters that is based on an Ir-catalyzed reductive Horner-Wadsworth-Emmons olefination. New β-ketotriene and pentaenethioates of pantetheine and N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.

Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-β1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-β1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-β1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-β1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-β1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-β1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-β1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-β1 to destabilize CFTR mRNA.

Streptomyces Cytochrome P450 Enzymes and Their Roles in the Biosynthesis of Macrolide Therapeutic Agents.

The study of the genus Streptomyces is of particular interest because it produces a wide array of clinically important bioactive molecules. The genomic sequencing of many Streptomyces species has revealed unusually large numbers of cytochrome P450 genes, which are involved in the biosynthesis of secondary metabolites. Many macrolide biosynthetic pathways are catalyzed by a series of enzymes in gene clusters including polyketide and non-ribosomal peptide synthesis. In general, Streptomyces P450 enzymes accelerate the final, post-polyketide synthesis steps to enhance the structural architecture of macrolide chemistry. In this review, we discuss the major Streptomyces P450 enzymes research focused on the biosynthetic processing of macrolide therapeutic agents, with an emphasis on their biochemical mechanisms and structural insights.

Plasticity in gene expression and fatty acid profiles of Acropora tenuis reciprocally transplanted between two water quality regimes in the central Great Barrier Reef, Australia

To investigate plasticity in biochemical and physiological health attributes of corals, identical colony fragments of the coral Acropora tenuis, from two inshore populations, were exposed to native and novel environmental regimes. Variation in global gene expression (GE) and lipid and fatty acid (FA) composition of surviving colony fragments were quantified after four months. Major FA classes, with the exception of short-chain (C18) polyunsaturated fatty acids (PUFA), decreased in concentration when coral fragments were exposed to lower water quality regardless of their source population. In contrast, a strong effect of source population was detected in the GE profiles of all coral fragments and was enriched with genes associated with translation, ribosome biogenesis and ribosome cellular components. One cluster of co-expressed genes positively correlated with multiple individual FA and included genes involved in developmental processes and cellular pathways. This study demonstrates the strong influence of a source effect defining gene expression relating to basic biological functions, including biosynthetic processing, translation and ribosome biogenesis. However, there is plasticity in FA composition and specific genes relating to elevated health and immunity, which can respond to changes in environmental conditions. These findings suggest hope for future corals, if we can reduce anthropogenic water quality stressors.

Single-Run Mass Spectrometry Analysis Provides Deep Insight into E. coli Proteome.

Single-run mass spectrometry has enabled the detection and quantifications of E. coli proteins. A total of 2068 proteins quantified by intensity-based absolute quantification (iBAQ) Schwanhäusser et al.: (Nature. 473, 337-342, 2011) procedure were obtained with single enzyme-trypsin, without pre-fractionation, by quadruplicate long liquid chromatography runs coupled with high-resolution linear trap quadrupole (LTQ)-Orbitrap Velos mass spectrometry. The single-run of 12h has ability to cover almost 98% of the quadruplicate LC-MS/MS runs of E. coli proteome and is therefore almost equivalent to quadruplicate LC-MS/MS runs. These quantified proteins are about 52% of the total proteins present in E. coli genome according to Uniprot database. The quantified proteins covered almost all of the proteins in folate biosynthesis. Remarkably greater part of Gene Ontology (GO) Barrell et al.: (Nucleic Acids Res. 37, D396-D403, 2009), Ashburner et al.: (Nat. Genet. 25, 25-29, 2000) annotations, signaling pathways along with protein-protein interactions were covered. Some of the important biological processes-cell cycle, DNA repair, ion transport, ubiquinone biosynthetic process, pseudouridine synthesis, peptidoglycan biosynthetic process, RNA processing, and translation-revealed protein-protein interaction network generated by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) Jensen, et al.:(Nucleic Acids Res 37, D412-D126, 2009) database. Therefore, to achieve the saturation point of detection of maximum number of proteins in single LC-MS/MS run, 12-h liquid chromatography gradient is appropriate. Graphical Abstract ᅟ.

Procollagen C-proteinase enhancer 1 (PCPE-1) functions as an anti-angiogenic factor and enhances epithelial recovery in injured cornea.

Procollagen C-proteinase enhancer 1 (PCPE-1) has been characterized as a protein capable of enhancing the activity of bone morphogenetic protein 1/tolloid-like proteinases in the biosynthetic processing of C-propeptides from procollagens I-III. This processing step is thought necessary to the formation of collagen I-III monomers capable of forming fibrils. Thus, PCPE-1 is predicted to play an important role in scarring, as scar tissue is predominantly composed of fibrillar collagen. Corneal scarring is of great clinical importance, as it leads to loss of visual acuity and, in severe cases, blindness. Here, we investigate a possible role for PCPE-1 in corneal scarring. Although differences in corneal opacity associated with scarring following injury of Pcolce -/- and wild-type (WT) mice using full-thickness excision or alkali burn models of corneal injury were not grossly apparent, differences in procollagen I processing levels between Pcolce -/- and WT primary corneal keratocytes were consistent with a role for PCPE-1 in corneal collagen deposition. An unexpected finding was that neoangiogenesis, which follows alkali burn cornea injury, was strikingly increased in Pcolce -/- cornea, compared to WT. A series of aortic ring assays confirmed the anti-angiogenic effects of PCPE-1. Another unexpected finding was of abnormalities of epithelial basement membrane and of re-epithelialization following Pcolce -/- corneal injury. Thus, PCPE-1 appears to be of importance as an anti-angiogenic factor and in re-epithelialization following injury in cornea and perhaps in other tissues as well.

Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-D-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

BMP1 and TLL1 Are Required for Maintaining Periodontal Homeostasis

Mutations in bone morphogenetic protein 1 (BMP1) in humans or deletion of BMP1 and related protease tolloid like 1 (TLL1) in mice lead to osteogenesis imperfecta (OI). Here, we show progressive periodontal defects in mice in which both BMP1 and TLL1 have been conditionally ablated, including malformed periodontal ligament (PDL) (recently shown to play key roles in normal alveolar bone formation), significant loss in alveolar bone mass (P < 0.01), and a sharp reduction in cellular cementum. Molecular mechanism studies revealed a dramatic increase in the uncleaved precursor of type I collagen (procollagen I) and a reduction in dentin matrix protein 1 (DMP1), which is partially responsible for defects in extracellular matrix (ECM) formation and mineralization. We also showed a marked increase in the expression of matrix metallopeptidase 13 (MMP13) and tartrate-resistant acid phosphatase (TRAP), leading to an acceleration in periodontal breakdown. Finally, we demonstrated that systemic application of antibiotics significantly improved the alveolar bone and PDL damage of the knockdown phenotype, which are thus shown to be partially secondary to pathogen-induced inflammation. Together, identification of the novel roles of BMP1 and TLL1 in maintaining homeostasis of periodontal formation, partly via biosynthetic processing of procollagen I and DMP1, provides novel insights into key contributions of the extracellular matrix environment to periodontal homeostasis and contributes toward understanding of the pathology of periodontitis.

Late-Exponential Gene Expression in codY-Deficient Bacillus anthracis in a Host-Like Environment.

CodY is a pleiotropic regulator commonly found in Gram-positive bacteria and regulates various biological processes during the stringent response in a nutrient-limiting environment. CodY also participates in virulence factor expression in many low G+C Gram-positive pathogens, as observed in Bacillus anthracis. However, the mechanism by which B. anthracis CodY regulates metabolism and virulence factors in response to environmental changes is unclear. Here, we attempted to identify the link between CodY and B. anthracis regulation with codY-deficient and codY-overexpressing mutants using high-throughput transcriptional analysis. Growth pattern analyses of codY mutants in both rich and minimal media showed defects in early cell proliferation, with opposite patterns in the early stationary phase: CodY overexpression prolonged bacterial growth, whereas deletion inhibited growth. RNA sequencing of codY-deficient B. anthracis showed both positive and negative changes in the gene expression of proteases and virulence factors as well as genes related to stringent response-related metabolism and biosynthetic processing. We also found that changes in codY expression could alter virulence gene expression of B. anthracis, suggesting modes of regulation in its virulence in a CodY concentration-dependent manner. Collectively, we conclude from these results that CodY can both positively and negatively regulate its regulon via direct and/or indirect approaches, and that its mode of regulation may be concentration dependent.

Functional characterisation of the type 1 von Willebrand disease candidate VWF gene variants: p.M771I, p.L881R and p.P1413L.

Abnormalities in the biosynthetic pathway or increased clearance of plasma von Willebrand factor (VWF) are likely to contribute to decreased plasma VWF levels in inherited type 1 von Willebrand disease (VWD). Recent studies demonstrated that 65% of type 1 VWD patients have candidate VWF mutations, the majority of which are missense variants. The purpose of this study was to explore the effects of three VWF missense mutations (p.M771I, p.L881R and p.P1413L) located in different functional domains of VWF, reported as candidate mutations in type 1 VWD patients in the course of the MCMDM-1VWD study. The focus of these studies was on the intracellular biosynthetic processing and localisation of VWF in a heterologous cell system. Molecular dynamic simulation for p.M771I and p.P1413L was also performed to analyse the conformational effects of the changes. As determined by immunofluorescence antibody staining and confocal microscopy of HEK293 cells, the intracellular localisation of recombinant VWF with the p.M771I variation was impaired. Transient transfection studies and phorbol myristate acetate stimulation in COS-7 cells revealed significant intracellular retention. In addition, major loss of VWF multimers was observed for only the p.M771I mutation. Molecular dynamic simulations on p.M771I mutant VWF revealed distinct structural rearrangements including a large deviation in the E' domain, and significant loss of β-sheet secondary structure. The pathogenic effects of candidate VWF gene mutations were explored in this study. In vitro expression studies in heterologous cell systems revealed impaired secretion of VWF and a dominant negative effect on the processing of the wild-type protein for only the p.M771I mutation and none of the mutations affected the regulated secretion.

BMP1-like proteinases are essential to the structure and wound healing of skin

Closely related extracellular metalloproteinases bone morphogenetic protein 1 (BMP1) and mammalian Tolloid-like 1 (mTLL1) are co-expressed in various tissues and have been suggested to have overlapping roles in the biosynthetic processing of extracellular matrix components. Early lethality of mice null for the BMP1 gene Bmp1 or the mTLL1 gene Tll1 has impaired in vivo studies of these proteinases. To overcome issues of early lethality and functional redundancy we developed the novel BTKO mouse strain, with floxed Bmp1 and Tll1 alleles, for induction of postnatal, simultaneous ablation of the two genes. We previously showed these mice to have a skeletal phenotype that includes elements of osteogenesis imperfecta (OI), osteomalacia, and deficient osteocyte maturation, observations validated by the finding of BMP1 mutations in a subset of human patients with OI-like phenotypes. However, the roles of BMP1-like proteinase in non-skeletal tissues have yet to be explored, despite the supposed importance of putative substrates of these proteinases in such tissues. Here, we employ BTKO mice to investigate potential roles for these proteinases in skin. Loss of BMP1-like proteinase activity is shown to result in markedly thinned and fragile skin with unusually densely packed collagen fibrils and delayed wound healing. We demonstrate deficits in the processing of collagens I and III, decorin, biglycan, and laminin 332 in skin, which indicate mechanisms whereby BMP1-like proteinases affect the biology of this tissue. In contrast, lack of effects on collagen VII processing or deposition indicates this putative substrate to be biosynthetically processed by non-BMP1-like proteinases.

PCSK1 Variants and Human Obesity.

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity.

Dissecting Reactions of Nonlinear Precursor Peptide Processing of the Class III Lanthipeptide Curvopeptin

Lanthipeptides are ribosomally synthesized peptides which undergo extensive post-translational modifications. In addition to novel structural features and bioactivities, the in vitro study on the biosynthesis of the class III lanthipeptide labyrinthopeptin revealed a unique C- to N-terminal directionality of biosynthetic processing. The recently described class III lanthipeptide curvopeptin allowed investigating the directionality aspect in much greater detail: Structural characterization of nine curvopeptin biosynthesis intermediates by high-resolution mass spectrometry combined with a deuterium-labeling approach enabled for the first time building a comprehensive biosynthesis model featuring all three post-translational modification reactions: phosphorylation, elimination, and cyclization. These results point to a nonlinear processing scheme with a predominant C → N-terminal directionality. Our data give important mechanistic insights into the concerted processing and directionality of the multifunctional class III modifying enzymes. The data are of significance in the light of obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of ribosomally synthesized and post-translationally modified peptides.

Tu1927 Genetically Driven Chromatin Organization Identifies Regulatory SNPs Associated With Crohn's Disease

Introduction: Genome-wide association studies suggest that there is dysregulation of distinct biological circuits involved in the maintenance of intestinal homeostasis in patients with inflammatory bowel diseases (IBD). Broadly, we seek to understand the relationship between patient genotype and disease-specific, tissue-dependent gene expression to elucidate critical pathways at play in health and disease, identify novel, clinically-applicable biomarkers, guide therapeutic discovery, and move towards personalized care for patients with IBD. Methods: Using the most-recent GWAS data for IBD, identifying 163 risk loci, a custom NanoString probeset of 683 IBD-associated genes and 15 housekeeping genes was designed; 25% of these genes were not assayed by conventional microarrays. Patients and controls were recruited at five IBD centers nationwide as part of the Sinai-Helmsley Alliance for Research Excellence (SHARE) consortium. To date, we have collected 352 specimens from 172 unique patients (Crohn's disease, n=142, ulcerative colitis, n=28, indeterminate colitis, n=2). Total mRNA was isolated from 2-3 mucosal biopsies obtained at colonoscopy or taken from surgical specimens and used for downstream processing. A custom bioanalytic pipeline was designed for quality control and normalization to permit differential expression, data-driven clustering, and genotype-based expression quantitative trait loci (eQTL) analysis. Results: To serve as a basis for analysis in patients, we defined the expression patterns of these selected genes in healthy controls in multiple anatomic locations, including terminal ileum, ascending and descending colon, observing distinct expression patterns between ileum and colon and high correlation between controls. In the second phase of this project, we compared the expression patterns between diseased tissue versus healthy controls, inflamed versus uninflamed tissues from the same patient, and ulcerative colitis versus Crohn's colitis. Preliminary analysis demonstrates a strong differential gene expression pattern with 120 upregulated genes and 8 down-regulated genes in the inflamed tissue as compared to the uninflamed tissue. Pathway analysis suggests differences in activity between inflamed and uninflamed tissues in pathways related to cell adhesion, immune system signaling, and biosynthetic processing. With targeted examination for eQTLs for four disease-related SNPs (NOD2, ATG16L1, IRGM, MST1), we have identified genotype-dependent, fold-change gene expression differences between paired inflamed and uninflamed specimens. Conclusions: Selected profiling of IBD-related genes in disease-relevant tissues by NanoString technology and in patients with known genotypes can robustly detect novel patterns of gene epistasis, identify eQTLs, and highlight dominant pathways perturbed in disease.

Tu1928 Gene Expression Alterations Suggest That Ulcerative Colitis Patients After Restorative Proctocolectomy Have Ileal Disease

Introduction: Genome-wide association studies suggest that there is dysregulation of distinct biological circuits involved in the maintenance of intestinal homeostasis in patients with inflammatory bowel diseases (IBD). Broadly, we seek to understand the relationship between patient genotype and disease-specific, tissue-dependent gene expression to elucidate critical pathways at play in health and disease, identify novel, clinically-applicable biomarkers, guide therapeutic discovery, and move towards personalized care for patients with IBD. Methods: Using the most-recent GWAS data for IBD, identifying 163 risk loci, a custom NanoString probeset of 683 IBD-associated genes and 15 housekeeping genes was designed; 25% of these genes were not assayed by conventional microarrays. Patients and controls were recruited at five IBD centers nationwide as part of the Sinai-Helmsley Alliance for Research Excellence (SHARE) consortium. To date, we have collected 352 specimens from 172 unique patients (Crohn's disease, n=142, ulcerative colitis, n=28, indeterminate colitis, n=2). Total mRNA was isolated from 2-3 mucosal biopsies obtained at colonoscopy or taken from surgical specimens and used for downstream processing. A custom bioanalytic pipeline was designed for quality control and normalization to permit differential expression, data-driven clustering, and genotype-based expression quantitative trait loci (eQTL) analysis. Results: To serve as a basis for analysis in patients, we defined the expression patterns of these selected genes in healthy controls in multiple anatomic locations, including terminal ileum, ascending and descending colon, observing distinct expression patterns between ileum and colon and high correlation between controls. In the second phase of this project, we compared the expression patterns between diseased tissue versus healthy controls, inflamed versus uninflamed tissues from the same patient, and ulcerative colitis versus Crohn's colitis. Preliminary analysis demonstrates a strong differential gene expression pattern with 120 upregulated genes and 8 down-regulated genes in the inflamed tissue as compared to the uninflamed tissue. Pathway analysis suggests differences in activity between inflamed and uninflamed tissues in pathways related to cell adhesion, immune system signaling, and biosynthetic processing. With targeted examination for eQTLs for four disease-related SNPs (NOD2, ATG16L1, IRGM, MST1), we have identified genotype-dependent, fold-change gene expression differences between paired inflamed and uninflamed specimens. Conclusions: Selected profiling of IBD-related genes in disease-relevant tissues by NanoString technology and in patients with known genotypes can robustly detect novel patterns of gene epistasis, identify eQTLs, and highlight dominant pathways perturbed in disease.