A structure and evolution-guided Monte Carlo sequence selection strategy for multiple alignment-based analysis of proteins

MOTIVATION: Various multiple sequence alignment-based methods have been proposed to detect functional surfaces in proteins, such as active sites or protein interfaces. The effect that the choice of sequences has on the conclusions of such analysis has seldom been discussed. In particular, no method has been discussed in terms of its ability to optimize the sequence selection for the reliable detection of functional surfaces. RESULTS: Here we propose, for the case of proteins with known structure, a heuristic Metropolis Monte Carlo strategy to select sequences from a large set of homologues, in order to improve detection of functional surfaces. The quantity guiding the optimization is the clustering of residues which are under increased evolutionary pressure, according to the sample of sequences under consideration. We show that we can either improve the overlap of our prediction with known functional surfaces in comparison with the sequence similarity criteria of selection or match the quality of prediction obtained through more elaborate non-structure based-methods of sequence selection. For the purpose of demonstration we use a set of 50 homodimerizing enzymes which were co-crystallized with their substrates and cofactors.     

Cbl promotes clustering of endocytic adaptor proteins

The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation.     

'Multifrequency' location and clustering of sequence patterns from proteins

In previous work, we have shown that a set of characteristics,defined as (code frequency) pairs, can be derived from a proteinfamily by the use of a signal-processing method. This methodenables the location and extraction of sequence patterns bytaking into account each (code frequency) pair individually.In the present paper, we propose to extend this method in orderto detect and visualize patterns by taking into account severalpairs simultaneously. Two ‘multifrequency’ methodsare described. The first one is based on a rewriting of thesequences with new symbols which summarize the frequency information.The second method is based on a clustering of the patterns associatedwith each pair. Both methods lead to the definition of significantconsensus sequences. Some results obtained with calcium-bindingproteins and serine proteases are also discussed. Received on March 6, 1990; accepted on September 24, 1990     

Neurodegeneration induced by clustering of sialylated glycosylphosphatidylinositols of prion proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.     

Functional clustering of yeast proteins from the protein-protein interaction network

Background  

The abundant data available for protein interaction networks have not yet been fully understood. New types of analyses are needed to reveal organizational principles of these networks to investigate the details of functional and regulatory clusters of proteins.     

Conformational response to charge clustering in synthetic intrinsically disordered proteins

Background

Recent theoretical and computational studies have shown that the charge content and, most importantly, the linear distribution of opposite charges are major determinants of conformational properties of intrinsically disordered proteins (IDPs). Charge segregation in a sequence can be measured through κ, which represents a normalized measure of charge asymmetry. A strong inverse correlation between κ and radius of gyration has been previously demonstrated for two independent sets of permutated IDP sequences.

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.     

Chromosomal clustering of nuclear genes encoding mitochondrial and chloroplast proteins in Arabidopsis

We present a statistical analysis of chromosomal clustering among nuclear genes encoding mitochondrial or chloroplast proteins in Arabidopsis. For both organelles, the clustering was significantly increased above the expectation, but the clustering effect was weak, and most clusters were small and dispersed. Clustered genes showed coexpression but not more than expected, and no substantial synteny was detected in other eukaryotic genomes. We propose that the unexpected clustering results from continuous selection favoring chromosomal proximity of genes acting in the same organelle.     

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.     

Hierarchical clustering of the correlation patterns: new method of domain identification in proteins

New method of identification of dynamical domains in proteins - Hierarchical Clustering of the Correlation Patterns (HCCP) is proposed. HCCP allows to identify the domains using single three-dimensional structure of the studied proteins and does not require any adjustable parameters that can influence the results. The method is based on hierarchical clustering performed on the matrices of correlation patterns, which are obtained by the transformation of ordinary pairwise correlation matrices. This approach allows to extract additional information from the correlation matrices, which increases reliability of domain identification. It is shown that HCCP is insensitive to small variations of the pairwise correlation matrices. Particularly it produces identical results if the data obtained for the same protein crystallized with different spatial positions of domains are used for analysis. HCCP can utilize correlation matrices obtained by any method such as normal mode or essential dynamics analysis, Gaussian network or anisotropic network models, etc. These features make HCCP an attractive method for domain identification in proteins.     

RIPCAL: a tool for alignment-based analysis of repeat-induced point mutations in fungal genomic sequences

Background  

Repeat-induced point mutation (RIP) is a fungal-specific genome defence mechanism that alters the sequences of repetitive DNA, thereby inactivating coding genes. Repeated DNA sequences align between mating and meiosis and both sequences undergo C:G to T:A transitions. In most fungi these transitions preferentially affect CpA di-nucleotides thus altering the frequency of certain di-nucleotides in the affected sequences. The majority of previously published in silico analyses were limited to the comparison of ratios of pre- and post-RIP di-nucleotides in putatively RIP-affected sequences – so-called RIP indices. The analysis of RIP is significantly more informative when comparing sequence alignments of repeated sequences. There is, however, a dearth of bioinformatics tools available to the fungal research community for alignment-based RIP analysis of repeat families.     

Functional clustering of metastasis proteins describes plastic adaptation resources of breast-cancer cells to new microenvironments

To examine the molecular mechanisms underlying breast cancer metastasis in liver and search for potential markers of metastatic progression in soft-tissue, we analyzed metastatic variants developed from the highly metastatic MDA-MB 435 cell line through in vivo stepwise selection in the athymic mice. Comparative proteomic analysis using two-dimensional electrophoresis (2DE-DIGE) revealed that 74 protein spots were reproducibly more than doubled in liver metastatic cells compared to parental counterpart. From 22 proteins identified by MALDI-TOF, belonging to intermediate filaments, intracellular transport and ATP synthesis, we generated a protein-protein interaction network containing 496 nodes, 12 of which interacted. GRP 75 was connected with four other proteins: prohibitin, HSP 27, elongin B and macropain delta chain. After functional classification, we found that pathways including hepatocyte growth factor receptor (p = 0.014), platelet-derived growth factor (p = 0.018), vascular endothelial growth factor (p = 0.021) and epidermal growth factor (p = 0.050) were predominant in liver metastatic cells, but not in lung metastatic cells. In conclusion, we suggest that GRP 75 is involved in cell proliferation, tumorigenesis and stress response in metastatic cells by recruiting signals in which the transmembrane receptor protein tyrosine kinase signaling pathway (p-value FDR = 1.71 x 10(-2)) and protein amino acid phosphorylation (p-value FDR = 3.28 x 10(-2)) might be the most significant biological process differentially increased in liver metastasis.     

Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions

Membrane proteins and membrane lipids are frequently organized in submicron-sized domains within cellular membranes. Factors thought to be responsible for domain formation include lipid-lipid interactions, lipid-protein interactions and protein-protein interactions. However, it is unclear whether the domain structure is regulated by other factors such as divalent cations. Here, we have examined in native plasma membranes and intact cells the role of the second messenger Ca(2+) in membrane protein organization. We find that Ca(2+) at low micromolar concentrations directly redistributes a structurally diverse array of membrane proteins via electrostatic effects. Redistribution results in a more clustered pattern, can be rapid and triggered by Ca(2+) influx through voltage-gated calcium channels and is reversible. In summary, the data demonstrate that the second messenger Ca(2+) strongly influences the organization of membrane proteins, thus adding a novel and unexpected factor that may control the domain structure of biological membranes.     

Functionally important segments in proteins dissected using Gene Ontology and geometric clustering of peptide fragments

We have developed a geometric clustering algorithm using backbone ,ψ angles to group conformationally similar peptide fragments of any length. By labeling each fragment in the cluster with the level-specific Gene Ontology 'molecular function' term of its protein, we are able to compute statistics for molecular function-propensity and p-value of individual fragments in the cluster. Clustering-cum-statistical analysis for peptide fragments 8 residues in length and with only trans peptide bonds shows that molecular function propensities ≥20 and p-values ≤0.05 can dissect fragments within a protein linked to the molecular function.     

Protein-DNA interactions: structural, thermodynamic and clustering patterns of conserved residues in DNA-binding proteins

Amino acid residues, which play important roles in protein function, are often conserved. Here, we analyze thermodynamic and structural data of protein-DNA interactions to explore a relationship between free energy, sequence conservation and structural cooperativity. We observe that the most stabilizing residues or putative hotspots are those which occur as clusters of conserved residues. The higher packing density of the clusters and available experimental thermodynamic data of mutations suggest cooperativity between conserved residues in the clusters. Conserved singlets contribute to the stability of protein-DNA complexes to a lesser extent. We also analyze structural features of conserved residues and their clusters and examine their role in identifying DNA-binding sites. We show that about half of the observed conserved residue clusters are in the interface with the DNA, which could be identified from their amino acid composition; whereas the remaining clusters are at the protein-protein or protein-ligand interface, or embedded in the structural scaffolds. In protein-protein interfaces, conserved residues are highly correlated with experimental residue hotspots, contributing dominantly and often cooperatively to the stability of protein-protein complexes. Overall, the conservation patterns of the stabilizing residues in DNA-binding proteins also highlight the significance of clustering as compared to single residue conservation.     

Hierarchical clustering based upon contextual alignment of proteins: a different way to approach phylogeny

We perform a computational study using a new approach to the analysis of protein sequences. The contextual alignment model, proposed recently by Gambin et al. (2002), is based on the assumption that, while constructing an alignment, the score of a substitution of one residue by another depends on the surrounding residues. The contextual alignment scores calculated in this model were used to hierarchical clustering of several protein families from the database of Clusters of Orthologous Groups (COG). The clustering has been also constructed based on the standard approach. The comparative analysis shows that the contextual model results in more consistent clustering trees. The difference, although small, is with no exception in favour of the contextual model. The consistency of the family of trees is measured by several consensus and agreement methods, as well as by the inter-tree distance approach.     

Implication of DNA polymerase lambda in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts

Accurate repair of free radical-mediated DNA double-strand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1- or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases mu and lambda, both of which were present in the extracts. For a DNA substrate with partially complementary 3' overhangs and a 2-base gap, antibodies to polymerase lambda completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase mu had little effect. Immunodepletion of polymerase lambda, but not polymerase mu, likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase lambda. Recombinant polymerase mu, and a truncated polymerase lambda lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase beta was completely inactive. The results suggest that polymerase lambda is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.