A fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis.

Mutations in the gene for fibrillin-1 cause Marfan syndrome (MFS), a common hereditary disorder of connective tissue. Recent findings suggest that proteolysis, increased matrix metalloproteinase activity, and fragmentation of fibrillin-rich microfibrils in tissues of persons with MFS contribute to the complex pathogenesis of this disorder. In this study we show that a fibrillin-1 fragment containing a EGFEPG sequence that conforms to a putative GxxPG elastin-binding protein (EBP) consensus sequence upregulates the expression and production of matrix metalloproteinase (MMP)-1 by up to ninefold in a cell culture system. A mutation of the GxxPG consensus sequence site abrogated the effects. This is the first demonstration of such an effect for ligands other than elastin fragments. Molecular dynamics analysis of oligopeptides with the wildtype and mutant sequence support our biochemical results by predicting significant alterations of structural characteristics such as the potential for forming a type VIII beta-turn that are thought to be important for binding to the EBP. These results suggest that fibrillin-1 fragments may regulate MMP-1 expression, and that the dysregulation of MMPs related to fragmentation of fibrillin might contribute to the development of MFS. Our Gene Ontology (GO) analysis of the human proteome shows that proteins with multiple GxxPG motifs are highly enriched for GO terms related to the extracellular matrix. Matrix proteins with multiple GxxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens. Some of these proteins have been associated with disorders involving alterations in MMP regulation, and the results of the present study suggest a potential mechanism for these observations.

Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region.

High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.

Workshop--Predicting the structure of biological molecules.

This April, in Cambridge (UK), principal investigators from the Mathematical Biology Group of the Medical Research Council's National Institute of Medical Research organized a workshop in structural bioinformatics at the Centre for Mathematical Sciences. Bioinformatics researchers of several nationalities from labs around the country presented and discussed their computational work in biomolecular structure prediction and analysis, and in protein evolution. The meeting was intensive and lively and gave attendees an overview of the healthy state of protein bioinformatics in the UK.

A review of bioinformatics education in the UK.

If the completion of the first draft of the human genome represents the coming of age of bioinformatics, then the emergence of bioinformatics as a university degree subject represents its establishment. In this paper bioinformatics as a subject for formal study is discussed, rather than as a subject for research, and a selection of the taught, mainly graduate, courses currently available in the UK are reviewed. Throughout, the author tries to draw parallels between the integration of bioinformatics into biomedical research and teaching today, and that of molecular biology, two decades ago. Others have made this analogy between these two relatively young disciplines. Although research sources are referenced, the author makes no pretence of objectivity. This article contains his opinions, and those of a number of current bioinformatics course organisers whose comments on the subject were solicited in advance specifically for this paper. The course organisers kindly advised how they planned their curricula, and described the special strengths of their programmes. Comments from present and former students of several bioinformatics degree programmes were also solicited. Except where individuals are directly quoted, any opinions expressed herein should be considered the author's. Compared with its sister piece [Marion Zatz, in previous issue of Briefings in Bioinformatics pp. 353], this paper is less about funding policy--which, in the UK, has lately (if belatedly) been more generous towards bioinformatics teaching--than it is about practice and content; the requirements of the bioinformatics research communities, the corresponding emphases of bioinformatics courses, and the general market for holders of bioinformatics degrees. Individual courses are cited throughout as examples, but the final section contains a full annotated listing with URL addresses. Based on the author's own experience of practising and teaching bioinformatics, he describes the skills he believes will be most useful to bioinformaticians in the near future and suggests ways to prepare students of bioinformatics for a fall in demand for those abilities.

Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy.

BACKGROUND: Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. METHODS AND RESULTS: We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with alpha-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to alpha-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. CONCLUSION: Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

Crystal structure of the CCTgamma apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin.

The chaperonin containing TCP-1 (CCT, also known as TRiC) is the only member of the chaperonin family found in the cytosol of eukaryotes. Like other chaperonins, it assists the folding of newly synthesised proteins. It is, however, unique in its specificity towards only a small subset of non-native proteins. We determined two crystal structures of mouse CCTgamma apical domain at 2.2 A and 2.8 A resolution. They reveal a surface patch facing the inside of the torus that is highly evolutionarily conserved and specific for the CCTgamma apical domain. This putative substrate-binding region consists of predominantly positively charged side-chains. It suggests that the specificity of this apical domain towards its substrate, partially folded tubulin, is conferred by polar and electrostatic interactions. The site and nature of substrate interaction are thus profoundly different between CCT and its eubacterial homologue GroEL, consistent with their different functions in general versus specific protein folding assistance.

Meeting review: 2002 O'Reilly Bioinformatics Technology Conference.

At the end of January I travelled to the States to speak at and attend the first O'Reilly Bioinformatics Technology Conference. It was a large, well-organized and diverse meeting with an interesting history. Although the meeting was not a typical academic conference, its style will, I am sure, become more typical of meetings in both biological and computational sciences.Speakers at the event included prominent bioinformatics researchers such as Ewan Birney, Terry Gaasterland and Lincoln Stein; authors and leaders in the open source programming community like Damian Conway and Nat Torkington; and representatives from several publishing companies including the Nature Publishing Group, Current Science Group and the President of O'Reilly himself, Tim O'Reilly. There were presentations, tutorials, debates, quizzes and even a 'jam session' for musical bioinformaticists.