Biomedical subjects

Peter Tompa

Publications and source records attributed to Peter Tompa.

28 records · Page 2Linked to original sources

PubMed2004

The role of structural disorder in the function of RNA and protein chaperones.

Chaperones are highly sophisticated protein machines that assist the folding of RNA molecules or other proteins. Their function is generally thought to require a fine-tuned and highly conserved structure: despite the recent recognition of the widespread occurrence of structural disorder in the proteome, this structural trait has never been generally considered in molecular chaperones. In this review we give evidence for the prevalence of functional regions without a well-defined 3-D structure in RNA and protein chaperones. By considering a variety of individual examples, we suggest that the structurally disordered chaperone regions either function as molecular recognition elements that act as solubilizers or locally loosen the structure of the kinetically trapped folding intermediate via transient binding to facilitate its conformational search. The importance of structural disorder is underlined by a predictor of natural disordered regions, which shows an extremely high proportion of such regions, unparalleled in any other protein class, within RNA chaperones: 54.2% of their residues fall into disordered regions and 40% fall within disordered regions longer than 30 consecutive residues. Structural disorder also prevails in protein chaperones, for which frequency values are 36.7% and 15%, respectively. In keeping with these and other details, a novel "entropy transfer" model is presented to account for the mechanistic role of structural disorder in chaperone function.

Animals↗

PubMed2004

Differential distribution of calpain small subunit 1 and 2 in rat brain.

Calpains, the Ca(2+)-dependent thiol proteases, are abundant in the nervous tissue. The ubiquitous enzyme forms in mammals are heterodimers consisting of a specific, micro or m, large (catalytic) subunit and, apparently, a common small (regulatory) subunit (CSS1). Recently, however, we described a second form of small subunit (CSS2), which is of restricted occurrence [Schád, E., Farkas, A., Jékely, G., Tompa, P. & Friedrich, P. (2002) Biochem. J., 362, 383-388]. Here we analysed the distribution of immunoreactivity in various parts of rat brain against two anti-CSS1 and two anti-CSS2 antibodies by correlated light and electron microscopy. Remarkably, the antibodies showed differential distribution in various parts of rat cortex: anti-CSS1 reacted mainly with perikarya and dendrites, whereas anti-CSS2 was more prominent in axons. In serial sections CSS2 and synaptophysin gave very similar patterns, i.e. these epitopes seem to colocalize. Electron microscopy confirmed that CSS1 was mainly localized postsynaptically in dendrites and somata, whereas CSS2 was found presynaptically. The hypothesis is advanced that these distinct distributions of calpain subunits may be related to the transport of these enzymes in nerve cells.

Animals↗

PubMed2003

Molecular cloning and RNA expression of a novel Drosophila calpain, Calpain C.

The calpains are Ca(2+)-activated cysteine proteases whose biochemical properties have been extensively characterized in vitro. Less is known, however, about the physiological role of calpains. In this respect, Drosophila melanogaster is a useful experimental organism to study calpain activity and regulation in vivo. The sequencing of the fly genome has been recently completed and a novel calpain homologue has been identified in the CG3692 gene product. We embarked on the cloning and characterization of this putative novel calpain. We demonstrate that the actual calpain is different from the predicted protein and we provide experimental evidence for the correction of the genomic annotation. This novel protein, Calpain C, must be catalytically inactive, having mutated active site residues but is otherwise structurally similar to the other known fly calpains. Moreover, we analysed Calpain C RNA expression during Drosophila development by RT-PCR and RNA in situ hybridization, which revealed strong expression in the salivary glands.

Amino Acid Sequence↗

PubMed2003

Intrinsically unstructured proteins evolve by repeat expansion.

The proportion of the genome encoding intrinsically unstructured proteins increases with the complexity of organisms, which demands specific mechanism(s) for generating novel genetic material of this sort. Here it is suggested that one such mechanism is the expansion of internal repeat regions, i.e., coding micro- and minisatellites. An analysis of 126 known unstructured sequences shows the preponderance of repeats: the percentage of proteins with tandemly repeated short segments is much higher in this class (39%) than earlier reported for all Swiss-Prot (14%), yeast (18%) or human (28%) proteins. Furthermore, prime examples, such as salivary proline-rich proteins, titin, eukaryotic RNA polymerase II, the prion protein and several others, demonstrate that the repetitive segments carry fundamental function in these proteins. In addition, their repeat numbers show functionally significant interspecies variation and polymorphism, which underlines that these regions have been shaped by intense evolutionary activity. In all, the major point of this paper is that the genetic instability of repetitive regions combined with the structurally and functionally permissive nature of unstructured proteins has powered the extension and possible functional expansion of this newly recognized protein class.

Algorithms↗

PubMed2003

Binding-induced folding transitions in calpastatin subdomains A and C.

Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.

Animals↗

PubMed2003

Revisiting ubiquity and tissue specificity of human calpains.

The abundance of mRNAs transcribed from human genes of the calpain superfamily was studied in 72 human tissues and cell types by the Human Multiple Tissue Expression (MTE) Array technique. The analysis included the large subunits of mu- and m-calpains, the small subunits, calpastatin and calpain 3 (p94). Besides specific data on transcriptional activity, two major conclusions emerged: (i) 'ubiquitous' calpains are not expressed in all cell types, and (ii) a 'tissue-specific' calpain may be expressed in many cell types apart from the one in which it is particularly abundant. Therefore, the categoric classification of 'ubiquitous' vs. 'tissue-specific' calpains is a simplification.

Calcium-Binding Proteins↗

PubMed2002

A novel human small subunit of calpains.

Typical calpains are heterodimeric cysteine proteases which have distinct large catalytic subunits (80 kDa) but share a common small regulatory subunit (30 kDa; css1). Here we report the identification, cloning and characterization of a novel human small subunit (css2) encoded by an intronless gene, capns2, located on chromosome 16. This new protein displays 73% sequence identity within the Ca(2+)-binding region but lacks two oligo-Gly stretches characteristic of the N-terminal domain of the conventional small subunit. css2 appears to be the functional equivalent of the conventional small subunit in vitro in that it helps the large subunit fold into the active conformation of similar Ca(2+) sensitivity when the two proteins are co-expressed in Escherichia coli. The purification of various chimaeric rat 80 kDa-human css2 constructs, on the other hand, shows that css2 binds the large subunit much more weakly than css1. Further, it does not undergo the autolytic conversion typical of the classical small subunit. The expression of this protein in vivo, as assessed from its appearance in expressed sequence tag clones, is rather limited, making it an example of a tissue-specific, rather than ubiquitous, small subunit.

Amino Acid Sequence↗

PubMed2002

Calpastatin subdomains A and C are activators of calpain.

The inhibitory domains of calpastatin contain three highly conserved regions, A, B, and C, of which A and C bind calpain in a strictly Ca(2+)-dependent manner but have no inhibitory activity whereas region B inhibits calpain on its own. We synthesized the 19-mer oligopeptides corresponding to regions A and C of human calpastatin domain I and tested their effect on human erythrocyte mu-calpain and rat m-calpain. The two peptides significantly activate both calpains: the Ca(2+) concentration required for half-maximal activity is lowered from 4.3 to 2.4 microm for mu-calpain and from 250 to 140 microm for m-calpain. The EC(50) concentration of the peptides is 7.5 microm for mu-calpain and 25 microm for m-calpain. It is noteworthy that at low Ca(2+) concentrations (1-2 microm for mu-calpain and 70-110 microm for m-calpain) both enzymes are activated about 10-fold by the peptides. Based on these findings, it is suggested that calpastatin fragments may have a role in calpain activation in vivo. Furthermore, these activators open new avenues to cell biological studies of calpain function and eventually may alleviate pathological states caused by calpain malfunction.

Amino Acid Sequence↗

PubMed2002

The role of dimerization in prion replication.

The central theme in prion diseases is the conformational transition of a cellular protein from a physiologic to a pathologic (so-called scrapie) state. Currently, two alternative models exist for the mechanism of this autocatalytic process; in the template assistance model the prion is assumed to be a monomer of the scrapie conformer, whereas in the nucleated polymerization model it is thought to be an amyloid rod. A recent variation on the latter assumes disulfide reshuffling as the mechanism of polymerization. The existence of stable dimers, let alone their mechanistic role, is not taken into account in either of these models. In this paper we review evidence supporting that the dimerization of either the normal or the scrapie state, or both, has a decisive role in prion replication. The contribution of redox changes, i.e., the temporary opening and possible rearrangement of the intramolecular disulfide bridge is also considered. We present a model including these features largely ignored so far and show that it adheres satisfactorily to the observed phenomenology of prion replication.

Animals↗

PubMed2002

Intrinsically unstructured proteins.

The recent suggestion that the classical structure-function paradigm should be extended to proteins and protein domains whose native and functional state is intrinsically unstructured has received a great deal of support. There is ample evidence that the unstructured state, common to all living organisms, is essential for basic cellular functions; thus it deserves to be recognized as a separate functional and structural category within the protein kingdom. In this review, recent findings are surveyed to illustrate that this novel but rapidly advancing field has reached a point where these proteins can be comprehensively classified on the basis of structure and function.

Amino Acid Sequence↗