REVCOM: a robust Bayesian method for evolutionary rate estimation

MOTIVATION: Evolutionary conservation estimated from a multiple sequence alignment is a powerful indicator of the functional significance of a residue and helps to predict active sites, ligand binding sites, and protein interaction interfaces. Many algorithms that calculate conservation work well, provided an accurate and balanced alignment is used. However, such a strong dependence on the alignment makes the results highly variable. We attempted to improve the conservation prediction algorithm by making it more robust and less sensitive to (1) local alignment errors, (2) overrepresentation of sequences in some branches and (3) occasional presence of unrelated sequences. RESULTS: A novel method is presented for robust constrained Bayesian estimation of evolutionary rates that avoids overfitting independent rates and satisfies the above requirements. The method is evaluated and compared with an entropy-based conservation measure on a set of 1494 protein interfaces. We demonstrated that approximately 62% of the analyzed protein interfaces are more conserved than the remaining surface at the 5% significance level. A consistent method to incorporate alignment reliability is proposed and demonstrated to reduce arbitrary variation of calculated rates upon inclusion of distantly related or unrelated sequences into the alignment.     

Protein synthesis-independent plasticity mediates rapid and precise recovery of deprived eye responses

Monocular deprivation (MD) for a few days during a critical period of development leads to loss of cortical responses to stimulation of the deprived eye. Despite the profound effects of MD on cortical function, optical imaging of intrinsic signals and single-unit recordings revealed that deprived eye responses and orientation selectivity recovered a few hours after restoration of normal binocular vision. Moreover, recovery of deprived eye responses was not dependent upon mRNA translation, but required cortical activity. Interestingly, this fast recovery and protein synthesis independence was restricted to the hemisphere contralateral to the previously deprived eye. Collectively, these results implicate a relatively simple mechanistic process in the reactivation of a latent set of connections following restoration of binocular vision and provide new insight into how recovery of cortical function can rapidly occur in response to changes in sensory experience.     

Primary cell cultures arising from normal kidney and renal cell carcinoma retain the proteomic profile of corresponding tissues

Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues.     

Expanding the proteome two-dimensional gel electrophoresis reference map of human renal cortex by peptide mass fingerprinting

Proteomics methodologies hold great promise in basic renal research and clinical nephrology. The classical approach for proteomic analysis couples two-dimensional gel electrophoresis (2-DE) with protein identification by mass spectrometry, to produce more global information regarding normal protein expression and alterations in different physiological and pathological states. In this report we have expanded the identification of proteins in the renal cortex, improving the previously published map to facilitate the study of different diseases affecting the human kidney. About 250 spots were analyzed by peptide mass fingerprinting, 89 proteins and 74 isoforms for some of them were identified and implemented in the normal human renal cortex 2-DE reference map. This more comprehensive view of the proteome of the human renal cortex could be of invaluable help to the differential proteomic display of urological diseases.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.     

Homeobox-clock protein interaction in zebrafish. A shared mechanism for pineal-specific and circadian gene expression

In non-mammalian vertebrates, the pineal gland is photoreceptive and contains an intrinsic circadian oscillator that drives rhythmic production and secretion of melatonin. These features require an accurate spatiotemporal expression of an array of specific genes in the pineal gland. Among these is the arylalkylamine N-acetyltransferase, a key enzyme in the melatonin production pathway. In zebrafish, pineal specificity of zfaanat2 is determined by a region designated the pineal-restrictive downstream module (PRDM), which contains three photoreceptor conserved elements (PCEs) and an E-box, elements that are generally associated with photoreceptor-specific and rhythmic expression, respectively. Here, by using in vivo and in vitro approaches, it was found that the PCEs and E-box of the PRDM mediate a synergistic effect of the photoreceptor-specific homeobox OTX5 and rhythmically expressed clock protein heterodimer, BMAL/CLOCK, on zfaanat2 expression. Furthermore, the distance between the PCEs and the E-box was found to be critical for PRDM function, suggesting a possible physical feature of this synergistic interaction. OTX5-BMAL/CLOCK may act through this mechanism to simultaneously control pineal-specific and rhythmic expression of zfaanat2 and possibly also other pineal and retinal genes.     

Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A

Heterotrimeric serine/threonine protein phosphatase 2A (PP2A) consists of scaffolding (A), catalytic (C), and variable (B, B', and B') subunits. Variable subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bbeta regulatory subunit gene is mutated in spinocerebellar ataxia type 12, and one of its splice variants, Bbeta2, targets PP2A to mitochondria to promote apoptosis in PC12 cells (Dagda, R. K., Zaucha, J. A., Wadzinski, B. E., and Strack, S. (2003) J. Biol. Chem. 278, 24976-24985). Here, we report that Bbeta2 is localized to the outer mitochondrial membrane by a novel mechanism, combining a cryptic mitochondrial import signal with a structural arrest domain. Scanning mutagenesis demonstrates that basic and hydrophobic residues mediate mitochondrial association and the proapoptotic activity of Bbeta2. When fused to green fluorescent protein, the N terminus of Bbeta2 acts as a cleavable mitochondrial import signal. Surprisingly, full-length Bbeta2 is not detectably cleaved and is retained at the outer mitochondrial membrane, even though it interacts with the TOM22 import receptor, as shown by luciferase complementation in intact cells. Mutations that open the C-terminal beta-propeller of Bbeta2 facilitate mitochondrial import, indicating that this rigid fold acts as a stop-transfer domain by resisting the partial unfolding step prerequisite for matrix translocation. Because hybrids of prototypical import and beta-propeller domains recapitulate this behavior, we predict the existence of other similarly localized proteins and a selection against highly stable protein folds in the mitochondrial matrix. This unfolding-resistant targeting to the mitochondrial translocase is necessary but not sufficient for the proapoptotic activity of Bbeta2, which also requires association with the rest of the PP2A holoenzyme.     

Cytogenetic Analysis of Experimental Hybrids in Species of Triatominae (Hemiptera-Reduviidae)

A cytogenetic analysis was performed in experimental hybrids between species of Chagas disease transmitting bugs with remarkable differences in the amount and distribution of heterochromatin. Using C-banding technique, we identified the parental species chromosomes and analysed the meiotic behaviour in the male hybrids between Triatoma platensis and T. infestans, T. platensis and T. delpontei, and T. infestans and T. rubrovaria. The two former hybrids have an entirely normal meiotic behaviour despite the extensive differences in C-banded karyotypes observed in the parental species, indicating that heterochromatin differences between homeologous chromosomes are not a barrier that influences meiotic synapsis and recombination. On the contrary, the experimental hybrids between T. infestans and T. rubrovaria show failures in pairing of homeologous chromosomes that lead to the production of abnormal spermatids and hybrid sterility. Our data suggest that karyotypic repatterning within triatomines has involved at least two different pathways. Among closely related species, chromosomal changes have largely involved addition or deletion of heterochromatic regions. In more distant species, chromosomal rearrangements (i.e. inversions and translocations) have also arisen. Hybridisation data also allow to hypothesize about the origin and divergence of this taxonomic group, as well as the mechanisms that maintain species isolation.     

Amino-terminally truncated Abeta peptide species are the main component of cotton wool plaques

Cotton wool plaques (CWPs) are round lesions that lack a central amyloid core. CWPs have been observed in individuals affected by early-onset familial Alzheimer disease (FAD) associated with mutations in the presenilin 1 (PSEN1) gene. Here we present the characterization of the amyloid-beta (Abeta) peptides deposited in the brain of an individual affected by FAD carrying the novel missense (V261I) mutation in the PSEN1 gene. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to determine the Abeta peptide species present in the cerebral and cerebellar cortices, in leptomeningeal vessels, and in CWPs isolated by laser microdissection (LMD). Our results indicate that amino-terminally truncated Abeta peptide species ending at residues 42 and 43 are the main Abeta peptides deposited in brain parenchyma and LMD-CWPs in association with the PSEN1 V261I mutation. Full-length Abeta1-42 and Abeta1-43 peptide species were underrepresented. CWPs were not found to be associated with vessels and did not contain Abeta1-40 peptides, the main component of the vascular deposits. Although Abeta deposits were present mostly in the form of CWPs in the cerebral cortex and as diffuse deposits in the cerebellar cortex, a similar array of amino-terminally truncated Abeta peptide species was seen in both cases. The biochemical data support the concept that parenchymal and vascular amyloid deposits are associated with a different array of Abeta peptide species. The generation and parenchymal deposition of highly insoluble amino-terminally truncated Abeta peptides may play an important role in the pathogenesis of AD and must be taken into consideration in developing new diagnostic and therapeutic strategies.