Integration of structural and functional genomics

This paper introduces a special issue of Animal Genetics , which is devoted to the recent symposium held at Iowa State University entitled 'Integration of Structural and Functional Genomics'. We describe issues and needs that confront the animal genomics community, and describe how this symposium was structured to address these issues by improving communication and collaboration across species and disciplines. The session topics and oral presentations are briefly described for all invited speakers.     

Thioredoxin - structural and functional complexity

Thioredoxins are small globular proteins that proved to be excellent model for investigating the relationship between the structure of protein and their physico-chemical and functional properties. The results from the experiments on thioredoxins offer the basic for the development of the new paradigms in the field of chemistry, biophysics and biology of proteins, with special attention to redox reaction in living cells, protein stability and design. It is a good example of broad class of sulphur-containing redox proteins.     

Protein structural alignments and functional genomics

Structural genomics-the systematic solution of structures of the proteins of an organism-will increasingly often produce molecules of unknown function with no close relative of known function. Prediction of protein function from structure has thereby become a challenging problem of computational molecular biology. The strong conservation of active site conformations in homologous proteins suggests a method for identifying them. This depends on the relationship between size and goodness-of-fit of aligned substructures in homologous proteins. For all pairs of proteins studied, the root-mean-square deviation (RMSD) as a function of the number of residues aligned varies exponentially for large common substructures and linearly for small common substructures. The exponent of the dependence at large common substructures is well correlated with the RMSD of the core as originally calculated by Chothia and Lesk (EMBO J 1986;5:823-826), affording the possibility of reconciling different structural alignment procedures. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins.     

Periplasmic chaperones--new structural and functional insights

Although chaperones exist in the periplasmic compartment of Gram-negative bacterial cells, how they function is not well understood. New intriguing functional insights are provided by the solved crystal structure of the periplasmic chaperone SurA.     

Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics

A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.     

Hodulcin: selective sweetness-reducing principle from Hovenia dulcis leaves

An aqueous extract of Hovenia dulcis leaves selectively reducedsweetness perception in humans. The taste-active principle,‘hodulcin’, was partially purified and comparedchromatographically with similarly prepared samples of the selective,sweetness-reducing compounds, gymnemic acids and ziziphins.Hodulcin appears to be a triterpene saponin glycoside, as arethe gymnemic acids and ziziphins. Nuclear magnetic resonance(NMR) spectra indicated an hodulcin aglycone structure differentfrom the gymnemic acids aglycone, and similar to, but not thesame as the ziziphins aglycone. Future comparative studies ofthe actions of hodulcin, gymnemic acids and ziziphins couldelucidate physiological mechanisms for the transduction andidentification of sweet stimuli and aid the development of newapproaches to the sweetening of foods.     

Human antiquitin: structural and functional studies

Antiquitin (ALDH7) is a member of the aldehyde dehydrogenase superfamily which oxidizes various aldehydes to form the corresponding carboxylic acids. Human antiquitin (ALDH7A1) is believed to play a role in detoxification, osmoregulation and more specifically, in lysine metabolism in which alpha-aminoadipic semialdehyde is identified as the specific, physiological substrate of the enzyme. In the present study, the structural basis for the substrate specificity was studied by site-directed mutagenesis. Kinetic analysis on wild-type human antiquitin and its mutants E121A and R301A demonstrated the importance of Glu121 and Arg301 in the binding as well as the turnover of alpha-aminoadipic semialdehyde. On the functional aspect, in addition to the already diversified physiological functions of antiquitin, the recent demonstration of its presence in the nucleus suggests that it may also play a role in cell growth and cell cycle progression. In this investigation, the expression level of antiquitin was monitored in synchronized WRL68 and HEK293 cell culture systems. It was found that the protein was up-regulated during G(1)-S phase transition. Immunofluorescence staining of the synchronized cells demonstrated an increased expression and accumulation of antiquitin in the nucleus during the G(1)-S phase transition. Knockdown of antiquitin using shRNA transfection also resulted in changes in the levels of several key cell cycle-regulating proteins.     

Blast sampling for structural and functional analyses

Background  

The post-genomic era is characterised by a torrent of biological information flooding the public databases. As a direct consequence, similarity searches starting with a single query sequence frequently lead to the identification of hundreds, or even thousands of potential homologues. The huge volume of data renders the subsequent structural, functional and evolutionary analyses very difficult. It is therefore essential to develop new strategies for efficient sampling of this large sequence space, in order to reduce the number of sequences to be processed. At the same time, it is important to retain the most pertinent sequences for structural and functional studies.     

Connexinopathies: a structural and functional glimpse

Mutations in human connexin (Cx) genes have been related to diseases, which we termed connexinopathies. Such hereditary disorders include nonsyndromic or syndromic deafness (Cx26, Cx30), Charcot Marie Tooth disease (Cx32), occulodentodigital dysplasia and cardiopathies (Cx43), and cataracts (Cx46, Cx50). Despite the clinical phenotypes of connexinopathies have been well documented, their pathogenic molecular determinants remain elusive. The purpose of this work is to identify common/uncommon patterns in channels function among Cx mutations linked to human diseases. To this end, we compiled and discussed the effect of mutations associated to Cx26, Cx32, Cx43, and Cx50 over gap junction channels and hemichannels, highlighting the function of the structural channel domains in which mutations are located and their possible role affecting oligomerization, gating and perm/selectivity processes.     

Connexinopathies: a structural and functional glimpse

Mutations in human connexin (Cx) genes have been related to diseases, which we termed connexinopathies. Such hereditary disorders include nonsyndromic or syndromic deafness (Cx26, Cx30), Charcot Marie Tooth disease (Cx32), occulodentodigital dysplasia and cardiopathies (Cx43), and cataracts (Cx46, Cx50). Despite the clinical phenotypes of connexinopathies have been well documented, their pathogenic molecular determinants remain elusive. The purpose of this work is to identify common/uncommon patterns in channels function among Cx mutations linked to human diseases. To this end, we compiled and discussed the effect of mutations associated to Cx26, Cx32, Cx43, and Cx50 over gap junction channels and hemichannels, highlighting the function of the structural channel domains in which mutations are located and their possible role affecting oligomerization, gating and perm/selectivity processes.

     

Antithrombin III: structural and functional aspects

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.     

Radiation induced structural and functional modification of haemoglobin

An abnormality in the primary structure of dog haemoglobin was observed 1-20 days after their whole body irradiation with 190 keV X-rays (4.0 Gy). It consisted in a substitution of tryptophane residue in position 15 of the beta-chain for serine. The percentage of abnormal beta-chains in different time intervals after irradiation was determined. The structural changes have functional impact: an increasing haemoglobin affinity to oxygen. This could be explained on the basis of changes in the tertiary structure of haemoglobin, which may result from the substitution Trp 15----Ser15 in the beta-chain which may influence the haeme ability to bind oxygen.     

Age-related structural and functional changes of brain mitochondria

Normal ageing is associated with a gradual decline in the capacity of various cell types, including neurones, to respond to metabolic stress and return to the resting state. An important factor in the decrease of this 'homeostatic reserve' is the gradual, age-dependent impairment of mitochondrial function. In this article we review some of the major structural and functional changes in mitochondria associated with ageing. Apart from the increased mutations in mitochondrial DNA and the evidence for increased oxidative stress with ageing, we also discuss, in some detail, the importance of the mitochondrial membrane structure and composition (in particular lipid composition) for mitochondrial function in general and during ageing. Although some of the neurodegenerative diseases are also associated with some degree of mitochondrial dysfunction, it is not yet clear if these changes are due to the underlining process of normal, physiological ageing or due to the specific pathophysiologic agents responsible for the neurodegenerative processes. Furthermore, we are proposing that there are important differences between normal ageing and neurodegeneration.     

Cholesterol and caveolae: structural and functional relationships

Caveolae are free cholesterol (FC)- and sphingolipid-rich surface microdomains abundant in most peripheral cells. Caveolin, a FC binding protein, is a major structural element of these domains. Caveolae serve as portals to regulate cellular FC homeostasis, possibly via their association with ancillary proteins including scavenger receptor B1. The FC content of caveolae regulates the transmission of both extracellular receptor-mediated and endogenous signal transduction via changes in the composition of caveolin-associated complexes of signaling intermediates. By controlling surface FC content, reporting membrane changes by signal transduction to the nucleus, and regulating signal traffic in response to extracellular stimuli, caveolae exert a multifaceted influence on cell physiology including growth and cell division, adhesion, and hormonal response. Cell surface lipid 'rafts' may assume many of the functions of caveolae in cells with low levels of caveolin.     

Storage and mobilization as antagonistic functional constraints on seed storage globulin evolution

When seeds germinate nearly all the proteins are degraded in senescing storage tissue cells. All these proteins act as amino acid reserves which are mobilized to nourish the seedling. Nevertheless, the major amount of the seeds' protein reserve consists of a few enzymatically inactive, abundant, genuine storage proteins. In their metabolism the conflicting processes of biosynthesis, protein turnover and breakdown, are temporally separated. No degradation of correctly formed storage proteins was observed at the time of synthesis and accumulation during seed maturation. Breakdown takes place after a (long) period of rest when seeds germinate and seedlings start growing. At that time genuine storage proteins are no longer synthesized. Genuine storage proteins have evolved structural features permitting controlled temporal patterns of protection and proteolysis. The acquisition of inserted sequence stretches as sites accessible to limited proteolysis played a key role in the evolution of this control system and happened in coevolution of genuine storage proteins with specific proteinases. This can be deduced from the results of current research on the mechanisms of limited and unlimited proteolysis of storage globulins and on storage globulin evolution. The evolved system of controlled structure-function interplay between storage globulins and proteinases is part of a syndrome that, in addition, comprises differential compartmentation and gene expression of storage proteins and proteinases for controlling the total spatial and temporal patterns of globulin storage and mobilization in maturing and germinating seeds.     

Oligomerization of H-pyrophosphatase and its structural and functional consequences

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.