Uros Midic

Unfoldomics of human diseases: linking protein intrinsic disorder with diseases

BackgroundIntrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) lack stable tertiary and/or secondary structure yet fulfills key biological functions. The recent recognition of IDPs and IDRs is leading to an entire field aimed at their systematic structural characterization and at determination of their mechanisms of action. Bioinformatics studies showed that IDPs and IDRs are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition and signal transduction. These activities complement the functions of structured proteins. IDPs and IDRs were shown to participate in both one-to-many and many-to-one signaling. Alternative splicing and posttranslational modifications are frequently used to tune the IDP functionality. Several individual IDPs were shown to be associated with human diseases, such as cancer, cardiovascular disease, amyloidoses, diabetes, neurodegenerative diseases, and others. This raises questions regarding the involvement of IDPs and IDRs in various diseases.ResultsIDPs and IDRs were shown to be highly abundant in proteins associated with various human maladies. As the number of IDPs related to various diseases was found to be very large, the concepts of the disease-related unfoldome and unfoldomics were introduced. Novel bioinformatics tools were proposed to populate and characterize the disease-associated unfoldome. Structural characterization of the members of the disease-related unfoldome requires specialized experimental approaches. IDPs possess a number of unique structural and functional features that determine their broad involvement into the pathogenesis of various diseases.ConclusionProteins associated with various human diseases are enriched in intrinsic disorder. These disease-associated IDPs and IDRs are real, abundant, diversified, vital, and dynamic. These proteins and regions comprise the disease-related unfoldome, which covers a significant part of the human proteome. Profound association between intrinsic disorder and various human diseases is determined by a set of unique structural and functional characteristics of IDPs and IDRs. Unfoldomics of human diseases utilizes unrivaled bioinformatics and experimental techniques, paves the road for better understanding of human diseases, their pathogenesis and molecular mechanisms, and helps develop new strategies for the analysis of disease-related proteins.

Protein disorder in the human diseasome: unfoldomics of human genetic diseases

BackgroundIntrinsically disordered proteins lack stable structure under physiological conditions, yet carry out many crucial biological functions, especially functions associated with regulation, recognition, signaling and control. Recently, human genetic diseases and related genes were organized into a bipartite graph (Goh KI, Cusick ME, Valle D, Childs B, Vidal M, et al. (2007) The human disease network. Proc Natl Acad Sci U S A 104: 8685–8690). This diseasome network revealed several significant features such as the common genetic origin of many diseases.Methods and findingsWe analyzed the abundance of intrinsic disorder in these diseasome network proteins by means of several prediction algorithms, and we analyzed the functional repertoires of these proteins based on prior studies relating disorder to function. Our analyses revealed that (i) Intrinsic disorder is common in proteins associated with many human genetic diseases; (ii) Different disease classes vary in the IDP contents of their associated proteins; (iii) Molecular recognition features, which are relatively short loosely structured protein regions within mostly disordered sequences and which gain structure upon binding to partners, are common in the diseasome, and their abundance correlates with the intrinsic disorder level; (iv) Some disease classes have a significant fraction of genes affected by alternative splicing, and the alternatively spliced regions in the corresponding proteins are predicted to be highly disordered; and (v) Correlations were found among the various diseasome graph-related properties and intrinsic disorder.ConclusionThese observations provide the basis for the construction of the human-genetic-disease-associated unfoldome.

Unfoldomics of human genetic diseases: illustrative examples of ordered and intrinsically disordered members of the human diseasome.

Intrinsically disordered proteins (IDPs) constitute a recently recognized realm of atypical biologically active proteins that lack stable structure under physiological conditions, but are commonly involved in such crucial cellular processes as regulation, recognition, signaling and control. IDPs are very common among proteins associated with various diseases. Recently, we performed a systematic bioinformatics analysis of the human diseasome, a network that linked the human disease phenome (which includes all the human genetic diseases) with the human disease genome (which contains all the disease-related genes) (Goh, K. I., Cusick, M. E., Valle, D., Childs, B., Vidal, M., and Barabasi, A. L. (2007). The human disease network. Proc. Natl. Acad. Sci. U.S.A. 104, 8685-90). The analysis of this diseasome revealed that IDPs are abundant in proteins linked to human genetic diseases, and that different genetic disease classes varied dramatically in the IDP content (Midic U., Oldfield C.J., Dunker A.K., Obradovic Z., Uversky V.N. (2009) Protein disorder in the human diseasome: Unfoldomics of human genetic diseases. BMC Genomics. In press). Furthermore, many of the genetic disease-related proteins were shown to contain at least one molecular recognition feature, which is a relatively short loosely structured protein region within a mostly disordered segment with the feature gaining structure upon binding to a partner. Finally, alternative splicing was shown to be abundant among the diseasome genes. Based on these observations the human-genetic-disease-associated unfoldome was created. This minireview describes several illustrative examples of ordered and intrinsically disordered members of the human diseasome.

Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants with the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals

In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. We find by cDNA microarray analysis enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. We also find that the key translational regulator, EIF4EBP1, undergoes a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appear at different positions along the spindle at different stages of meiosis. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types.

Extensive effects of in vitro oocyte maturation on rhesus monkey cumulus cell transcriptome

The elaboration of a quality oocyte is integrally linked to the correct developmental progression of cumulus cell phenotype. In humans and nonhuman primates, oocyte quality is diminished with in vitro maturation. To determine the changes in gene expression in rhesus monkey cumulus cells (CC) that occur during the final day prior to oocyte maturation and how these changes differ between in vitro (IVM) and in vivo maturation (VVM), we completed a detailed comparison of transcriptomes using the Affymetrix gene array. We observed a large number of genes differing in expression when comparing IVM-CC and VVM-CC directly but a much larger number of differences when comparing the transitions from the prematuration to the post-IVM and post-VVM states. We observed a truncation or delay in the normal pattern of gene regulation but also remarkable compensatory changes in gene expression during IVM. Among the genes affected by IVM are those that contribute to productive cell-cell interactions between cumulus cell and oocyte and between cumulus cells. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not to be suppressed during IVM. We identified a panel of 24 marker genes, the expression of which should provide the foundation for understanding how IVM can be improved for monitoring IVM conditions and for diagnosing oocyte quality.

Transgenerational effects of binge drinking in a primate model: implications for human health

OBJECTIVE To determine if binge ethanol consumption before ovulation affects oocyte quality, gene expression, and subsequent embryo development. DESIGN Binge levels of ethanol were given twice weekly for 6 months, followed by a standard in vitro fertilization cycle and subsequent natural mating. SETTING National primate research center. ANIMAL(S) Adult female rhesus monkeys. INTERVENTION(S) Binge levels of ethanol, given twice weekly for 6 months before a standard in vitro fertilization cycle with or without embryo culture. With in vivo development, ethanol treatment continued until pregnancy was identified. MAIN OUTCOME MEASURE(S) Oocyte and cumulus/granulosa cell gene expression, embryo development to blastocyst, and pregnancy rate. RESULT(S) Embryo development in vitro was reduced; changes were found in oocyte and cumulus cell gene expression; and spontaneous abortion during very early gestation increased. CONCLUSION(S) This study provides evidence that binge drinking can affect the developmental potential of oocytes even after alcohol consumption has ceased.

Systems Genetics Implicates Cytoskeletal Genes in Oocyte Control of Cloned Embryo Quality

Cloning by somatic cell nuclear transfer is an important technology, but remains limited due to poor rates of success. Identifying genes supporting clone development would enhance our understanding of basic embryology, improve applications of the technology, support greater understanding of establishing pluripotent stem cells, and provide new insight into clinically important determinants of oocyte quality. For the first time, a systems genetics approach was taken to discover genes contributing to the ability of an oocyte to support early cloned embryo development. This identified a primary locus on mouse chromosome 17 and potential loci on chromosomes 1 and 4. A combination of oocyte transcriptome profiling data, expression correlation analysis, and functional and network analyses yielded a short list of likely candidate genes in two categories. The major category—including two genes with the strongest genetic associations with the traits (Epb4.1l3 and Dlgap1)—encodes proteins associated with the subcortical cytoskeleton and other cytoskeletal elements such as the spindle. The second category encodes chromatin and transcription regulators (Runx1t1, Smchd1, and Chd7). Smchd1 promotes X chromosome inactivation, whereas Chd7 regulates expression of pluripotency genes. Runx1t1 has not been associated with these processes, but acts as a transcriptional repressor. The finding that cytoskeleton-associated proteins may be key determinants of early clone development highlights potential roles for cytoplasmic components of the oocyte in supporting nuclear reprogramming. The transcriptional regulators identified may contribute to the overall process as downstream effectors.

Intrinsic disorder in putative protein sequences

BackgroundIntrinsically disordered proteins (IDPs) and regions (IDRs) perform a variety of crucial biological functions despite lacking stable tertiary structure under physiological conditions in vitro. State-of-the-art sequence-based predictors of intrinsic disorder are achieving per-residue accuracies over 80%. In a genome-wide study of intrinsic disorder in human genome we observed a big difference in predicted disorder content between confirmed and putative human proteins. We investigated a hypothesis that this discrepancy is not correct, and that it is due to incorrectly annotated parts of the putative protein sequences that exhibit some similarities to confirmed IDRs, which lead to high predicted disorder content.MethodsTo test this hypothesis we trained a predictor to discriminate sequences of real proteins from synthetic sequences that mimic errors of gene finding algorithms. We developed a procedure to create synthetic peptide sequences by translation of non-coding regions of genomic sequences and translation of coding regions with incorrect codon alignment.ResultsApplication of the developed predictor to putative human protein sequences showed that they contain a substantial fraction of incorrectly assigned regions. These regions are predicted to have higher levels of disorder content than correctly assigned regions. This partially, albeit not completely, explains the observed discrepancy in predicted disorder content between confirmed and putative human proteins.ConclusionsOur findings provide the first evidence that current practice of predicting disorder content in putative sequences should be reconsidered, as such estimates may be biased.

Protein sequence alignment and structural disorder: a substitution matrix for an extended alphabet

In protein sequence alignment algorithms, a substitution matrix of 20x20 alignment parameters is used to describe the rates of amino acid substitutions over time. Development and evaluation of most substitution matrices including the BLOSUM family [1] was based almost entirely on fully structured proteins. Structurally disordered proteins (i.e. proteins that lack structure, either in part or as a whole) that have been shown to be very common in nature [2] have a significantly different amino acid composition than ordered (i.e. structured) proteins [3]. Furthermore, the sequence evolution rate is higher in unstructured as compared to structured regions of proteins containing both structured and unstructured regions [4]. These results cast doubt on appropriateness of the BLOSUM substitution matrices for alignment of structurally disordered proteins [5].To address this problem, we take into the account the concept of structural disorder by extending the alphabet for sequence representation from 20 to 2x20=40 symbols, 20 for amino acids in disordered regions and 20 for amino acids in ordered regions. A 40x40 substitution matrix is required for alignment of sequences represented in the extended alphabet. Such an expanded matrix contains 20x20 submatrices that correspond to matching ordered-ordered, ordered-disordered, and disordered-disordered pairs of residues. In this paper we describe an iterative procedure that we used to estimate such a 40x40 substitution matrix. The iterative procedure converged with stable results with respect to the choice of the sequences in the dataset. In the obtained 40x40 matrix we found substantial differences between the 20x20 submatrices corresponding to ordered-ordered, ordered-disordered, and disordered-disordered region matching. These differences provide evidence that for alignment of protein sequences that contain disordered segments, the discovered substitution matrix is more appropriate than the BLOSUM substitution matrices. At the same time, the new substitution matrix is applicable for sequence alignment of fully ordered proteins as its order-order submatrix is very similar to a BLOSUM matrix.

Improving Protein Secondary-Structure Prediction by Predicting Ends of Secondary-Structure Segments

Motivated by known preferences for certain amino acids in positions around a-helices, we developed neural network-based predictors of both N and C a-helix ends, which achieved about 88% accuracy. We applied a similar approach for predicting the ends of three types of secondary structure segments. The predictors for the ends of H, E and C segments were then used to create input for protein secondary-structure prediction. By incorporating this new type of input, we significantly improved the basic one-stage predictor of protein secondary structure in terms of both per-residue (Q3) accuracy (+0.8%) and segment overlap (SOV3) measure (+1.4).