Lajos Kalmár

TOPDB: topology data bank of transmembrane proteins

The Topology Data Bank of Transmembrane Proteins (TOPDB) is the most complete and comprehensive collection of transmembrane protein datasets containing experimentally derived topology information currently available. It contains information gathered from the literature and from public databases available on the internet for more than a thousand transmembrane proteins. TOPDB collects details of various experiments that were carried out to learn about the topology of particular transmembrane proteins. In addition to experimental data from the literature, an extensive collection of structural data was also compiled from PDB and from PDBTM. Because topology information is often incomplete, for each protein in the database the most probable topology that is consistent with the collected experimental constraints was also calculated using the HMMTOP transmembrane topology prediction algorithm. Each record in TOPDB also contains information on the given protein sequence, name, organism and cross references to various other databases. The web interface of TOPDB includes tools for searching, relational querying and data browsing as well as for visualization. TOPDB is designed to bridge the gap between the number of transmembrane proteins available in sequence databases and the publicly accessible topology information of experimentally or computationally studied transmembrane proteins. TOPDB is available at http://topdb.enzim.hu.

Structural disorder in proteins brings order to crystal growth in biomineralization

Biomineralization, the generation of hard tissues of living organisms, is a process strictly regulated by hormones, enzymes and a range of regulatory proteins of which several resisted structural characterization thus far. Without actual generalizations, there have been scattered observations in the literature for the structural disorder of these proteins. To address this issue in general, we have collected SwissProt proteins involved in the formation of bone and teeth in vertebrates, annotated for biomineralization. All these proteins show an extremely high level of predicted disorder (with a mean of 53%), making them the most disordered functional class of the protein world. Exactly the same feature was established for evolutionarily more distant proteins involved in the formation of the silica wall of marine diatoms and the shell of oysters and other mollusks. Because these proteins also show an extremely biased amino acid composition, such as high negative charge, high frequency of Ser and Asp or Pro residues and repetitiveness, we also carried out a database search with these sequence features for further proteins. This search uncovered several further disordered proteins with clearly related functions, although their annotations made no mention of biomineralization. This general and very strong correlation between biomineralization, structural disorder of proteins and particular sequence features indicates that regulated growth of mineral phase in biology can only be achieved by the assistance of highly disordered proteins.

Narcolepsy patients have antibodies that stain distinct cell populations in rat brain and influence sleep patterns

Significance Narcolepsy is a chronic sleep disease with autoimmune origin. We explored occurrence of autoantibodies in narcolepsy and other sleep-related disorders (OSRDs) by screening human sera with immunohistochemistry on rat brains. Hypocretin/orexinergic neurons were not stained, but a prominent immunostaining pattern of hypothalamic melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) neurons was overrepresented in cases of narcolepsy and OSRD patients. The autoantigen was identified as the common C-terminal epitope of neuropeptide glutamic acid-isoleucine/α–melanocyte-stimulating hormone (NEI/αMSH). Purified IgGs from a patient with MCH/POMC staining injected intracerebroventricularly to rats caused disturbed sleep patterns. Also, GABAergic cortical interneurons were stained with other narcolepsy and OSRD sera. Thus, autoantibodies are frequent in patients with sleep disorders, and NEI/αMSH may be a previously unidentified autoantigen involved in pathomechanism(s). These findings indicate possible diagnostic/therapeutic targets. Narcolepsy is a chronic sleep disorder, likely with an autoimmune component. During 2009 and 2010, a link between A(H1N1)pdm09 Pandemrix vaccination and onset of narcolepsy was suggested in Scandinavia. In this study, we searched for autoantibodies related to narcolepsy using a neuroanatomical array: rat brain sections were processed for immunohistochemistry/double labeling using patient sera/cerebrospinal fluid as primary antibodies. Sera from 89 narcoleptic patients, 52 patients with other sleep-related disorders (OSRDs), and 137 healthy controls were examined. Three distinct patterns of immunoreactivity were of particular interest: pattern A, hypothalamic melanin-concentrating hormone and proopiomelanocortin but not hypocretin/orexin neurons; pattern B, GABAergic cortical interneurons; and pattern C, mainly globus pallidus neurons. Altogether, 24 of 89 (27%) narcoleptics exhibited pattern A or B or C. None of the patterns were exclusive for narcolepsy but were also detected in the OSRD group at significantly lower numbers. Also, some healthy controls exhibited these patterns. The antigen of pattern A autoantibodies was identified as the common C-terminal epitope of neuropeptide glutamic acid-isoleucine/α–melanocyte-stimulating hormone (NEI/αMSH) peptides. Passive transfer experiments on rat showed significant effects of pattern A human IgGs on rapid eye movement and slow-wave sleep time parameters in the inactive phase and EEG θ-power in the active phase. We suggest that NEI/αMSH autoantibodies may interfere with the fine regulation of sleep, contributing to the complex pathogenesis of narcolepsy and OSRDs. Also, patterns B and C are potentially interesting, because recent data suggest a relevance of those brain regions/neuron populations in the regulation of sleep/arousal.

Reduction in structural disorder and functional complexity in the thermal adaptation of prokaryotes.

Genomic correlates of evolutionary adaptation to very low or very high optimal growth temperature (OGT) values have been the subject of many studies. Whereas these provided a protein-structural rationale of the activity and stability of globular proteins/enzymes, the point has been neglected that adaptation to extreme temperatures could also have resulted from an increased use of intrinsically disordered proteins (IDPs), which are resistant to these conditions in vitro. Contrary to these expectations, we found a conspicuously low level of structural disorder in bacteria of very high (and very low) OGT values. This paucity of disorder does not reflect phylogenetic relatedness, i.e. it is a result of genuine adaptation to extreme conditions. Because intrinsic disorder correlates with important regulatory functions, we asked how these bacteria could exist without IDPs by studying transcription factors, known to harbor a lot of function-related intrinsic disorder. Hyperthermophiles have much less transcription factors, which have reduced disorder compared to their mesophilic counterparts. On the other hand, we found by systematic categorization of proteins with long disordered regions that there are certain functions, such as translation and ribosome biogenesis that depend on structural disorder even in hyperthermophiles. In all, our observations suggest that adaptation to extreme conditions is achieved by a significant functional simplification, apparent at both the level of the genome and individual genes/proteins.

Dual coding in alternative reading frames correlates with intrinsic protein disorder

Numerous human genes display dual coding within alternatively spliced regions, which give rise to distinct protein products that include segments translated in more than one reading frame. To resolve the ensuing protein structural puzzle, we identified 67 human genes with alternative splice variants comprising a dual-coding region at least 75 nucleotides in length and analyzed the structural status of the protein segments they encode. The inspection of their amino acid composition and predictions by the IUPred and PONDR® VSL2 algorithms suggest a high propensity for structural disorder in dual-coding regions. In the case of +1 frameshifts, the average level of disorder in the two frames is similarly high (47.2% in the ancestral frame, 58.2% in the derived frame, with the average level of disorder in human proteins being approximately 30%), whereas in the case of -1 frameshifts, there is a significant tendency to become more disordered upon shifting the frame (16.7% in the ancestral frame, 56.3% in the derived frame). The regions encoded by the derived frame are mostly disordered (disorder percentage > 50%) in 39 out of 62 cases, which strongly suggests that structural disorder enables these protein products to exist and function without the need of a highly evolved 3D fold. The potential advantages are also demonstrated by the appearance of novel functions and the high incidence of transcripts escaping nonsense-mediated decay. By discussing several examples, we demonstrate that dual coding may be an effective mechanism for the evolutionary appearance of novel intrinsically disordered regions with new functions.

Verification of alternative splicing variants based on domain integrity, truncation length and intrinsic protein disorder

According to current estimations ∼95% of multi-exonic human protein-coding genes undergo alternative splicing (AS). However, for 4000 human proteins in PDB, only 14 human proteins have structures of at least two alternative isoforms. Surveying these structural isoforms revealed that the maximum insertion accommodated by an isoform of a fully ordered protein domain was 5 amino acids, other instances of domain changes involved intrinsic structural disorder. After collecting 505 minor isoforms of human proteins with evidence for their existence we analyzed their length, protein disorder and exposed hydrophobic surface. We found that strict rules govern the selection of alternative splice variants aimed to preserve the integrity of globular domains: alternative splice sites (i) tend to avoid globular domains or (ii) affect them only marginally or (iii) tend to coincide with a location where the exposed hydrophobic surface is minimal or (iv) the protein is disordered. We also observed an inverse correlation between the domain fraction lost and the full length of the minor isoform containing the domain, possibly indicating a buffering effect for the isoform protein counteracting the domain truncation effect. These observations provide the basis for a prediction method (currently under development) to predict the viability of splice variants.

The P28T Mutation in the GALK1 Gene Accounts for Galactokinase Deficiency in Roma (Gypsy) Patients across Europe

Galactokinase deficiency is an inborn error of metabolism that, if untreated, results in the development of cataracts in the first weeks of life. The disorder is rare worldwide, but has a high incidence among the Roma (Gypsies). In 1999, we reported the founder Romani mutation, P28T, identified in affected families from Bulgaria. Subsequent studies have detected the same mutation in Romani patients from different European countries. The screening of 803 unrelated control individuals of Romani ethnicity from Bulgaria, Hungary, and Spain has shown an overall carrier rate of 1:47 and an expected incidence of affected births about 1:10,000. Using disease haplotype analysis, the age of the P28T mutation was estimated at 750 y, preceding the splits of the proto-Roma into the numerous populations resident in Europe today. The findings suggest that the mutation has spread with the early diaspora of the Roma throughout Europe. Superimposed on this old distribution pattern is the new migration wave of the last decade, with large numbers of Roma moving to Western Europe as a result of the economic changes in the East and the wars in former Yugoslavia. The changing demographic pattern of Romani minorities can be expected to lead to a homogenization of the incidence of “private” Romani disorders and founder mutations. The P28T mutation is thus likely to account for a high proportion of galactokinase deficiency cases across Europe. Mutation-based pilot newborn screening programs would provide current incidence figures and help to design long-term prevention of infantile cataracts due to galactokinase deficiency.

TOPDOM: database of domains and motifs with conservative location in transmembrane proteins.

Summary: The TOPDOM database is a collection of domains and sequence motifs located consistently on the same side of the membrane in α-helical transmembrane proteins. The database was created by scanning well-annotated transmembrane protein sequences in the UniProt database by specific domain or motif detecting algorithms. The identified domains or motifs were added to the database if they were uniformly annotated on the same side of the membrane of the various proteins in the UniProt database. The information about the location of the collected domains and motifs can be incorporated into constrained topology prediction algorithms, like HMMTOP, increasing the prediction accuracy. Availability: The TOPDOM database and the constrained HMMTOP prediction server are available on the page http://topdom.enzim.hu Contact: tusi@enzim.hu; lkalmar@enzim.hu

The other side of the coin: Functional and structural versatility of ADF/cofilins

Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in the regulation of this cellular machinery is the ADF-H domain proteins. Despite sharing the same domain, ADF-H domain proteins produce a diverse functional landscape in the regulation of the actin cytoskeleton. Recent findings emphasize that the functional and structural features of these proteins can differ not only between ADF-H families but even within the same family. The structural and evolutional background of this functional diversity is poorly understood. This review focuses on the specific functional characteristics of ADF-H domain proteins and how these features can be linked to structural differences in the ADF-H domain and also to different conformational transitions in actin. In the light of recent discoveries we pay special attention to the ADF/cofilin proteins to find tendencies along which the functional and structural diversification is governed through the evolution.

Power Law Distribution Defines Structural Disorder as a Structural Element Directly Linked with Function

Although intrinsically disordered proteins are prevalent and functionally important, it has never been asked whether structural disorder should be considered as a separate structural category on its own or merely as a lack of secondary and/or tertiary structure. We address this issue by showing that its length distribution in the human proteome follows a power law, with many short regions but also a significant incidence of very long disordered regions. This behavior is in sharp contrast with that of conventional secondary structural elements and is highly reminiscent of the distribution of tertiary structural units in proteins. We interpret this finding by the direct functional involvement of disorder, which distinguishes it from secondary structural elements and endows it with tertiary structural attributes.