Biomedical subjects

Michal Linial

Publications and source records attributed to Michal Linial.

33 records · Page 2Linked to original sources

PubMed2004

A robust method to detect structural and functional remote homologues.

With currently available sequence data, it is feasible to conduct extensive comparisons among large sets of protein sequences. It is still a much more challenging task to partition the protein space into structurally and functionally related families solely based on sequence comparisons. The ProtoNet system automatically generates a treelike classification of the whole protein space. It stands to reason that this classification reflects evolutionary relationships, both close and remote. In this article, we examine this hypothesis. We present a semiautomatic procedure that singles out certain inner nodes in the ProtoNet tree that should ideally correspond to structurally and functionally defined protein families. We compare the performance of this method against several expert systems. Some of the competing methods incorporate additional extraneous information on protein structure or on enzymatic activities. The ProtoNet-based method performs at least as well as any of the methods with which it was compared. This article illustrates the ProtoNet-based method on several evolutionarily diverse families. Using this new method, an evolutionary divergence scheme can be proposed for a large number of structural and functional related superfamilies.

Computational Biology↗

PubMed2004

Predicting fold novelty based on ProtoNet hierarchical classification.

MOTIVATION: Structural genomics projects aim to solve a large number of protein structures with the ultimate objective of representing the entire protein space. The computational challenge is to identify and prioritize a small set of proteins with new, currently unknown, superfamilies or folds. RESULTS: We develop a method that assigns each protein a likelihood of it belonging to a new, yet undetermined, structural superfamily. The method relies on a variant of ProtoNet, an automatic hierarchical classification scheme of all protein sequences from SwissProt. Our results show that proteins that are remote from solved structures in the ProtoNet hierarchy are more likely to belong to new superfamilies. The results are validated against SCOP releases from recent years that account for about half of the solved structures known to date. We show that our new method and the representation of ProtoNet are superior in detecting new targets, compared to our previous method using ProtoMap classification. Furthermore, our method outperforms PSI-BLAST search in detecting potential new superfamilies.

Algorithms↗

PubMed2004

Evolutional insights on uncharacterized SARS coronavirus genes.

The complete genome of the severe acute respiratory syndrome coronavirus (SARS-CoV) and many of its variants has been determined by several laboratories. The genome contains fourteen predicted open reading frames (ORFs). However, a function had been clearly assigned for only six of these ORFs, in the viral replication, transcription and structural constituents. The others are herein referred to as uncharacterized ORFs (UC-ORFs). Here, we try to provide a relational insight on those UC-ORFs, suggesting that a number of them are remotely related to structural proteins of coronaviruses and other viruses infecting mammalian hosts. Surprisingly, several of the UC-ORFs exhibit considerable similarity with other SARS-CoV ORFs. These observations may provide clues on the evolution and genome dynamics of the SARS-CoV.

Amino Acid Sequence↗

PubMed2004

Hydrophobized dextran-spermine conjugate as potential vector for in vitro gene transfection.

Dextran polysaccharide was grafted by reductive-amination with mixtures of spermine and other natural/synthetic oligoamines of two to four amine groups. The transfection efficiencies of the polycations thus obtained were assessed in various cell lines, and found to depend on the spermine contents. Higher spermine ratios of grafted oligoamines resulted in high gene expression, whereas low to negligible expressions were obtained with lower spermine contents. The effect was explained by spermine residues which exhibit altered buffering capacity in comparison to other substituted oligoamines. Hydrophobization of dextran-spermine (D-SPM) was achieved by treating the polymer with N-hydroxysuccinimide derivatives of cholesterol and fatty acids in a mixture of water/THF. The degree of hydrophobization was in the range of 1-30% mol/mol (hydrophobic moieties/primary amine) and the coupling yields were >95% as determined by (1)H-NMR. The oleate-modified D-SPM remarkably enhanced the gene expression in serum rich media, in marked contrast to unmodified D-SPM which resulted with a drastic decrease in the transfection yields. Modified D-SPM derivatives of other fatty acids and cholesterol showed improved transfection yields in comparison to unmodified D-SPM, but to a lower extent when compared to oleate modification. The improvement in cell transfection was attributed to oleate residues which probably play a role in increasing stability and uptake of polycation-DNA complexes.

Amines↗

PubMed2003

PANDORA: keyword-based analysis of protein sets by integration of annotation sources.

Recent advances in high-throughput methods and the application of computational tools for automatic classification of proteins have made it possible to carry out large-scale proteomic analyses. Biological analysis and interpretation of sets of proteins is a time-consuming undertaking carried out manually by experts. We have developed PANDORA (Protein ANnotation Diagram ORiented Analysis), a web-based tool that provides an automatic representation of the biological knowledge associated with any set of proteins. PANDORA uses a unique approach of keyword-based graphical analysis that focuses on detecting subsets of proteins that share unique biological properties and the intersections of such sets. PANDORA currently supports SwissProt keywords, NCBI Taxonomy, InterPro entries and the hierarchical classification terms from ENZYME, SCOP and GO databases. The integrated study of several annotation sources simultaneously allows a representation of biological relations of structure, function, cellular location, taxonomy, domains and motifs. PANDORA is also integrated into the ProtoNet system, thus allowing testing thousands of automatically generated clusters. We illustrate how PANDORA enhances the biological understanding of large, non-uniform sets of proteins originating from experimental and computational sources, without the need for prior biological knowledge on individual proteins.

Computational Biology↗

PubMed2003

Direct interaction of target SNAREs with the Kv2.1 channel. Modal regulation of channel activation and inactivation gating.

Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.

Animals↗

PubMed2003

ProtoNet: hierarchical classification of the protein space.

The ProtoNet site provides an automatic hierarchical clustering of the SWISS-PROT protein database. The clustering is based on an all-against-all BLAST similarity search. The similarities' E-score is used to perform a continuous bottom-up clustering process by applying alternative rules for merging clusters. The outcome of this clustering process is a classification of the input proteins into a hierarchy of clusters of varying degrees of granularity. ProtoNet (version 1.3) is accessible in the form of an interactive web site at http://www.protonet.cs.huji.ac.il. ProtoNet provides navigation tools for monitoring the clustering process with a vertical and horizontal view. Each cluster at any level of the hierarchy is assigned with a statistical index, indicating the level of purity based on biological keywords such as those provided by SWISS-PROT and InterPro. ProtoNet can be used for function prediction, for defining superfamilies and subfamilies and for large-scale protein annotation purposes.

Animals↗

PubMed2003

How incorrect annotations evolve--the case of short ORFs.

The draft of the human genome sequence is still incomplete. The outstanding tasks include filling in some gaps, finalizing the assembly of short sequences, improving sequence accuracy and correctly identifying coding regions. However, a closely related problem that receives little attention is the substantial number of incorrect annotations that have penetrated some of the widely used databases. This article illustrates this problem using the example of ubiquitin genes, and draws some conclusions that apply to false annotations in other short open reading frames (ORFs). Although the focus is on the human genome, other genomes are equally prone to similar propagation of false annotations.

Databases, Nucleic Acid↗

PubMed2003

PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells.

Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.

Animals↗

PubMed2002

Polysaccharide-oligoamine based conjugates for gene delivery.

This work describes a versatile and universal polycation system based on oligoamines grafted on natural polysaccharides that is capable of complexing various plasmids and administering them into various cells in high yield to produce a desired protein. These polycations are expected to better meet the requirements for effective complexation and delivery of plasmid or an antisense and to biodegrade into nontoxic components at a controlled rate. The developed biodegradable polycations are based on spermine, a natural tetramine, conjugated to dextran or arabinogalactan. These polycations were prepared by reductive amination of oxidized polysaccharides with the desired oligoamines. The Schiff base conjugates thus obtained were reduced to the stable amine conjugates by sodium borohydride. Over 300 different polycations were prepared starting from various polysaccharides and oligoamines, mainly oligoamines of two to four amino groups. Although most of these conjugates formed stable complexes with various plasmids as determined by turbidity experiments, only a few polycations were found to be active in transfecting cells. This work indicates that the structure of the polycation plays a significant role in the transfection activity of polycations.

Cations↗

PubMed2002

The advantage of functional prediction based on clustering of yeast genes and its correlation with non-sequence based classifications.

Sequence similarity is probably the most widely used tool to infer functional linkage between proteins. The fully sequenced, much researched, genome of Saccharomyces cerevisiae gives us on opportunity to compare and statistically quantify computational methods based on sequence similarity, which aim to detect such linkage. In addition, the amount of data regarding Saccharomyces Cerevisiae genes and proteins, which is not directly based on sequence is rapidly increasing. Consequently, it allows investigation of the connections and correlation between classification based on these types of data and that based solely on sequence similarity. In this work we start with a simple clustering algorithm to cluster genes based on the BLAST E-score of their similarity. We analyze how well one can infer function from these clusters and for how many of the genes that are currently unknown one can suggest a prediction. Given these parameters, we show that even a simple algorithm achieves better results than simply considering the BLAST output of matching genes. In the second part of the paper, we show that there is a highly significant correlation (p-value < 10(-4) for the vast majority of the experiments) between the aforementioned clusters and other types of classifications. Namely, we show that a pair of genes being clustered together is correlated with these genes having similar expression patterns in DNA array experiments and with the encoded proteins being involved in protein-protein interactions. Although this correlation is highly significant, it is, of course, not strong enough to be, by itself, a tool for predicting co-regulation of genes or interaction of proteins. We discuss possible explanations for this correlation. Furthermore, the statistical evaluation of these results should be considered when developing tools that are aimed at making such predictions.

Algorithms↗

PubMed2002

Selecting targets for structural determination by navigating in a graph of protein families.

MOTIVATION: A major goal in structural genomics is to enrich the catalogue of proteins whose 3D structures are known. In an attempt to address this problem we mapped over 10 000 proteins with solved structures onto a graph of all Swissprot protein sequences (release 36, approximately 73 000 proteins) provided by ProtoMap, with the goal of sorting proteins according to their likelihood of belonging to new superfamilies. We hypothesized that proteins within neighbouring clusters tend to share common structural superfamilies or folds. If true, the likelihood of finding new superfamilies increases in clusters that are distal from other solved structures within the graph. RESULTS: We defined an order relation between unsolved proteins according to their 'distance' from solved structures in the graph, and sorted approximately 48 000 proteins. Our list can be partitioned into three groups: approximately 35 000 proteins sharing a cluster with at least one known structure; approximately 6500 proteins in clusters with no solved structure but with neighbouring clusters containing known structures; and a third group contains the rest of the proteins, approximately 6100 (in 1274 clusters). We tested the quality of the order relation using thousands of recently solved structures that were not included when the order was defined. The tests show that our order is significantly better (P-value approximately 10(5)) than a random order. More interestingly, the order within the union of the second and third groups, and the order within the third group alone, perform better than random (P-values: 0.0008 and 0.15, respectively) and are better than alternative orders created using PSI-BLAST. Herein, we present a method for selecting targets to be used in structural genomics projects. AVAILABILITY: List of proteins to be used for targets selection combined with a set of biological filters for narrowing down potential targets is in http://www.protarget.cs.huji.ac.il.

Algorithms↗

PubMed2002

The metric space of proteins-comparative study of clustering algorithms.

MOTIVATION: A large fraction of biological research concentrates on individual proteins and on small families of proteins. One of the current major challenges in bioinformatics is to extend our knowledge to very large sets of proteins. Several major projects have tackled this problem. Such undertakings usually start with a process that clusters all known proteins or large subsets of this space. Some work in this area is carried out automatically, while other attempts incorporate expert advice and annotation. RESULTS: We propose a novel technique that automatically clusters protein sequences. We consider all proteins in SWISSPROT, and carry out an all-against-all BLAST similarity test among them. With this similarity measure in hand we proceed to perform a continuous bottom-up clustering process by applying alternative rules for merging clusters. The outcome of this clustering process is a classification of the input proteins into a hierarchy of clusters of varying degrees of granularity. Here we compare the clusters that result from alternative merging rules, and validate the results against InterPro. Our preliminary results show that clusters that are consistent with several rather than a single merging rule tend to comply with InterPro annotation. This is an affirmation of the view that the protein space consists of families that differ markedly in their evolutionary conservation.

Algorithms↗

PubMed2002

Pardaxin stimulation of phospholipases A2 and their involvement in exocytosis in PC-12 cells.

Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A(2) inhibitors we show that PX stimulation of phospholipase A(2) (PLA(2)) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA(2) activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E(2) (PGE(2)), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE(2), and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE(2) induced by PX. In the absence of extracellular calcium, the calcium-independent PLA(2) (iPLA(2)) inhibitors methyl arachidonyl fluorophosphonate, AACOCF(3), and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE(2) release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented by N-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE(2), and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA(2) and iPLA(2), leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release.

Animals↗

PubMedDate not supplied

Quaternary ammonium polysaccharides for gene delivery.

Cationic polysaccharides were synthesized by conjugation of various monoquaternary (MQ) ammonium oligoamines to oxidized dextran by reductive amination and tested for gene transfection. Polycations of dextran grafted with MQ ammonium oligoamines of two to four amino groups were investigated for their ability to cause pCMV-GFP encoding for green fluorescence protein and beta-Gal encoding for beta-galactosidase protein transfection on EPC and HEK-293 cell lines. These polycations were expected to strongly complex DNA due to increased surface cationic charge of the carrier, which may result in a higher transfection yield. However, the transfection yields were much lower compared to the parent vector, dextran-spermine conjugate, which was highly effective both in vitro and in vivo.

Cell Line↗