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Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the alpha2(I) chain of type I collagen and cleaves the three alpha chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into 3 ⁄4 and 1 ⁄4 fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25°C. The results indicate that the nine residues 183 RWTNNFREY191 located between the fifth ␤-strand and the second ␣-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr191 of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.Interstitial collagen types I, II, and III are the major structural proteins in connective tissues such as tendon, skin, bone, cartilage, and blood vessels. They consist of three ␣ chains with repeating Gly-X-Y triplets where X and Y are frequently Pro and Hyp, respectively. Each chain of the repeating tripeptide adopts a left-handed poly-Pro II helix conformation, and three left-handed chains then intertwine to form a right-handed superhelix (1-3). This triple helical conformation makes interstitial collagens resistant to most proteinases in vertebrates except for collagenases, cathepsin K (4), and neutrophil elastase (5). The action of cathepsin K is probably important in collagen breakdown in specialized environments such as during bone resorption in an acidic pH environment. Neutrophil elastase may degrade telopeptides of interstitial collagen (6) and the triple-helical region of type I collagen under inflammatory conditions, but the latter activity is much weaker than that of collagenase (5). Vertebrate collagenases, on the other hand, are synthesized by many cell types such as stromal fibroblasts, chondrocytes, keratinocytes, osteoblasts, endothelial cells, and macrophages in response to inflammatory cytokines, growth factors, cellular transformation, and other chemical and physical stimuli ...

An important component of precision medicine—the use of whole-genome sequencing (WGS) to guide lifelong healthcare—is electronic decision support to inform drug choice and dosing. To achieve this, automated identification of genetic variation in genes involved in drug absorption, distribution, metabolism, excretion and response (ADMER) is required. CYP2D6 is a major enzyme for drug bioactivation and elimination. CYP2D6 activity is predominantly governed by genetic variation; however, it is technically arduous to haplotype. Not only is the nucleotide sequence of CYP2D6 highly polymorphic, but the locus also features diverse structural variations, including gene deletion, duplication, multiplication events and rearrangements with the nonfunctional, neighbouring CYP2D7 and CYP2D8 genes. We developed Constellation, a probabilistic scoring system, enabling automated ascertainment of CYP2D6 activity scores from 2×100 paired-end WGS. The consensus reference method included TaqMan genotyping assays, quantitative copy-number variation determination and Sanger sequencing. When compared with the consensus reference Constellation had an analytic sensitivity of 97% (59 of 61 diplotypes) and analytic specificity of 95% (116 of 122 haplotypes). All extreme phenotypes, i.e., poor and ultrarapid metabolisers were accurately identified by Constellation. Constellation is anticipated to be extensible to functional variation in all ADMER genes, and to be performed at marginal incremental financial and computational costs in the setting of diagnostic WGS.

Genomics has contributed to a growing collection of gene-function and gene-disease annotations that can be exploited by informatics to study similarity between diseases. This can yield insight into disease etiology, reveal common pathophysiology and/or suggest treatment that can be appropriated from one disease to another. Estimating disease similarity solely on the basis of shared genes can be misleading as variable combinations of genes may be associated with similar diseases, especially for complex diseases. This deficiency can be potentially overcome by looking for common biological processes rather than only explicit gene matches between diseases. The use of semantic similarity between biological processes to estimate disease similarity could enhance the identification and characterization of disease similarity. We present functions to measure similarity between terms in an ontology, and between entities annotated with terms drawn from the ontology, based on both co-occurrence and information content. The similarity measure is shown to outperform other measures used to detect similarity. A manually curated dataset with known disease similarities was used as a benchmark to compare the estimation of disease similarity based on gene-based and Gene Ontology (GO) process-based comparisons. The detection of disease similarity based on semantic similarity between GO Processes (Recall=55%, Precision=60%) performed better than using exact matches between GO Processes (Recall=29%, Precision=58%) or gene overlap (Recall=88% and Precision=16%). The GO-Process based disease similarity scores on an external test set show statistically significant Pearson correlation (0.73) with numeric scores provided by medical residents. GO-Processes associated with similar diseases were found to be significantly regulated in gene expression microarray datasets of related diseases.

BackgroundDNA methylation of CpG sites on genetic loci has been linked to increased risk of asthma in children exposed to elevated ambient air pollutants (AAPs). Further identification of specific CpG sites and the pollutants that are associated with methylation of these CpG sites in immune cells could impact our understanding of asthma pathophysiology. In this study, we sought to identify some CpG sites in specific genes that could be associated with asthma regulation (Foxp3 and IL10) and to identify the different AAPs for which exposure prior to the blood draw is linked to methylation levels at these sites. We recruited subjects from Fresno, California, an area known for high levels of AAPs. Blood samples and responses to questionnaires were obtained (n = 188), and in a subset of subjects (n = 33), repeat samples were collected 2 years later. Average measures of AAPs were obtained for 1, 15, 30, 90, 180, and 365 days prior to each blood draw to estimate the short-term vs. long-term effects of the AAP exposures.ResultsAsthma was significantly associated with higher differentially methylated regions (DMRs) of the Foxp3 promoter region (p = 0.030) and the IL10 intronic region (p = 0.026). Additionally, at the 90-day time period (90 days prior to the blood draw), Foxp3 methylation was positively associated with NO2, CO, and PM2.5 exposures (p = 0.001, p = 0.001, and p = 0.012, respectively). In the subset of subjects retested 2 years later (n = 33), a positive association between AAP exposure and methylation was sustained. There was also a negative correlation between the average Foxp3 methylation of the promoter region and activated Treg levels (p = 0.039) and a positive correlation between the average IL10 methylation of region 3 of intron 4 and IL10 cytokine expression (p = 0.030).ConclusionsShort-term and long-term exposures to high levels of CO, NO2, and PM2.5 were associated with alterations in differentially methylated regions of Foxp3. IL10 methylation showed a similar trend. For any given individual, these changes tend to be sustained over time. In addition, asthma was associated with higher differentially methylated regions of Foxp3 and IL10.Electronic supplementary materialThe online version of this article (10.1186/s13148-017-0433-4) contains supplementary material, which is available to authorized users.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity.

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