Connectivity of Neutral Networks,Overdispersion, and Structural Conservation in Protein Evolution

Abstract Protein structures are much more conserved than sequences during evolution. Based on this observation, we investigate the consequences of structural conservation on protein evolution. We study seven of the most studied protein folds, determining that an extended neutral network in sequence space is associated with each of them. Within our model, neutral evolution leads to a non-Poissonian substitution process, due to the broad distribution of connectivities in neutral networks. The observation that the substitution process has non-Poissonian statistics has been used to argue against the original Kimura neutral theory, while our model shows that this is a generic property of neutral evolution with structural conservation. Our model also predicts that the substitution rate can strongly fluctuate from one branch to another of the evolutionary tree. The average sequence similarity within a neutral network is close to the threshold of randomness, as observed for families of sequences sharing the same fold. Nevertheless, some positions are more difficult to mutate than others. We compare such structurally conserved positions to positions conserved in protein evolution, suggesting that our model can be a valuable tool to distinguish structural from functional conservation in databases of protein families. These results indicate that a synergy between database analysis and structurally based computational studies can increase our understanding of protein evolution.     

Targeting of proteins to the twin-arginine translocation pathway

The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N-terminal signal sequence containing a highly conserved twin-arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.     

Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution.

Three-dimensional structures have been elucidated for very few integral membrane proteins. Computer methods can be used as guides for estimation of solute transport protein structure, function, biogenesis, and evolution. In this paper the application of currently available computer programs to over a dozen distinct families of transport proteins is reviewed. The reliability of sequence-based topological and localization analyses and the importance of sequence and residue conservation to structure and function are evaluated. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific transport protein families is also evaluated. Channel proteins are proposed to be functionally related to carriers. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is shown that several transport protein families have evolved independently of each other, employing different routes, at different times in evolutionary history, to give topologically similar transmembrane protein complexes. The possible significance of this apparent topological convergence is discussed.     

Novel mechanisms for the targeting of proteins into and across chloroplast membranes

The assembly of the photosynthetic apparatus requires the translocation of numerous proteins from the cytosol, initially into the stroma and thereafter into or across the thylakoid membrane. Recent studies have shown that proteins are transported into this membrane by a variety of mechanisms, some of which are derived from a cyanobacterial-type ancestor, whereas others have evolved in response to the more complex transport pathway used by cytosolically synthesized chloroplast proteins. It is now apparent that some of the targeting pathways are used exclusively by hydrophobic thylakoid membrane proteins; here we review recent progress in our understanding of the biogenesis of this important class of protein.     

Protein-protein interaction network-based detection of functionally similar proteins within species

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.     

Phylogenetic characterization of novel transport protein families revealed by genome analyses

As a result of recent genome sequencing projects as well as detailed biochemical, molecular genetic and physiological experimentation on representative transport proteins, we have come to realize that all organisms possess an extensive but limited array of transport protein types that allow the uptake of nutrients and excretion of toxic substances. These proteins fall into phylogenetic families that presumably reflect their evolutionary histories. Some of these families are restricted to a single phylogenetic group of organisms and may have arisen recently in evolutionary time while others are found ubiquitously and may be ancient. In this study we conduct systematic phylogenetic analyses of 26 families of transport systems that either had not been characterized previously or were in need of updating. Among the families analyzed are some that are bacterial-specific, others that are eukaryotic-specific, and others that are ubiquitous. They can function by either a channel-type or a carrier-type mechanism, and in the latter case, they are frequently energized by coupling solute transport to the flux of an ion down its electrochemical gradient. We tabulate the currently sequenced members of the 26 families analyzed, describe the properties of these families, and present partial multiple alignments, signature sequences and phylogenetic trees for them all.     

The Opisthokonta and the Ecdysozoa may not be clades: stronger support for the grouping of plant and animal than for animal and fungi and stronger support for the Coelomata than Ecdysozoa

In considering the best possible solutions for answering phylogenetic questions from genomic sequences, we have chosen a strategy that we suggest is superior to others that have gone previously. We have ignored multigene families and instead have used single-gene families. This minimizes the inadvertent analysis of paralogs. We have employed strict data controls and have reasoned that if a protein is not capable of recovering the uncontroversial parts of a phylogenetic tree, then why should we use it for the more controversial parts? We have sliced and diced the data in as many ways as possible in order to uncover the signals in that data. Using this strategy, we have tested two controversial hypotheses concerning eukaryotic phylogenetic relationships: the placement of arthropoda and nematodes and the relationships of animals, plants, and fungi. We have constructed phylogenetic trees from 780 single-gene families from 10 completed genomes and amalgamated these into a single supertree. We have also carried out a total evidence analysis on the only universally distributed protein families that can accurately reconstruct the uncontroversial parts of the phylogenetic tree: a total of five families. In doing so, we ignore the majority of single-gene families that are universally distributed as they do not have the appropriate signals to recover the uncontroversial parts of the tree. We have also ignored every protein that has ever been used previously to address this issue, simply because none of them meet our strict criteria. Using these data controls, site stripping, and multiple analyses, 24 out of 26 analyses strongly support the grouping of vertebrates with arthropods (Coelomata hypothesis) and plants with animals. In the other two analyses, the data were ambivalent. The latter finding overturns an 11-year theory of Eukaryotic evolution; the first confirms what has already been said by others. In the light of this new tree, we re-analyze the evolution of intron gain and loss in the rpL14 gene and find that it is much more compatible with the hypothesis presented here than with the Opisthokonta hypothesis.     

Managing the manganese: molecular mechanisms of manganese transport and homeostasis

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.     

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.     

Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B(0) transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B(0)-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes of invertebrate and vertebrate organisms, outlining a new possibility for selective targeting of essential amino acid absorption mechanisms to control medically and economically important arthropods and other invertebrate organisms.     

Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle

Macromolecular transport between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC) and is mediated by multiple families of soluble transport factors. All these transport factors share the ability to translocate across the NPC through specific interactions with components of the nuclear pore. This review highlights advances in our understanding of the structure and function of the NPC and the shuttling transport receptors involved in nuclear transport. It discusses recently proposed models for the translocation of receptor-cargo complexes through the NPC channel and reviews how the small GTPase Ran functions as a positional marker of the genome to regulate multiple important aspects of the eukaryotic cell cycle.     

Latent periodicity of protein families, identified with the indel-aware algorithm

Latent amino acid repeats seem to be widespread in genetic sequences and to reflect their structure, function, and evolution. We have recently identified latent periodicity in more than 150 protein families including protein kinases and various nucleotide-binding proteins. The latent repeats in these families were correlated to their structure and evolution. However, a majority of known protein families were not identified with our latent periodicity search algorithm. The main presumable reason for this was the inability of our techniques to identify periodicities interspersed with insertions and deletions. We designed the new latent periodicity search algorithm, which is capable of taking into account insertions and deletions. As a result, we identified many novel cases of latent periodicity peculiar to protein families. Possible origins of the periodic structure of these families are discussed. Summarizing, we presume that latent periodicity is present in a substantial portion of known protein families. The latent periodicity matrices and the results of Swiss-Prot scans are available from http://bioinf.narod.ru/del/.     

Nucleocytoplasmic transport: cargo trafficking across the border

Transport of macromolecules between the cytoplasm and the nucleus is mediated by at least three different classes of soluble transport receptors, members of the importin-beta protein family, the Mex67/Tap family and the small nuclear transport factor 2 (NFT2). All nuclear transport factors can bidirectionally traverse the nuclear pore complex through specific interactions with phenylalanine/glycine-rich nuclear pore complex components. Recent kinetic and structural analyses revealed novel insight into the details of these interactions. In addition, new biochemical and genetic studies have dramatically improved our understanding of ribosomal and messenger RNA export, unveiling a tight coupling between RNA processing and transport.     

Exiting the endoplasmic reticulum

Vesicular transport from the endoplasmic reticulum (ER) to the Golgi complex constitutes the initial step in protein secretion. COPII-coated vesicles mediate the export of newly synthesized proteins from the ER, and this transport step is coupled with COPI-mediated retrograde traffic to form a transport circuit that supports the compositional asymmetry of the ER-Golgi system. Biochemical and structural studies have advanced our understanding of the mechanisms that control vesicle formation and cargo-protein capture. Recent work has highlighted the function of transitional ER regions in specifying the location of COPII budding.     

Microbial oceanography: paradigms, processes and promise

Life on Earth most likely originated as microorganisms in the sea. Over the past approximately 3.5 billion years, microorganisms have shaped and defined Earth's biosphere and have created conditions that have allowed the evolution of macroorganisms and complex biological communities, including human societies. Recent advances in technology have highlighted the vast and previously unknown genetic information that is contained in extant marine microorganisms, from new protein families to novel metabolic processes. Now there is a unique opportunity, using recent advances in molecular ecology, metagenomics, remote sensing of microorganisms and ecological modelling, to achieve a comprehensive understanding of marine microorganisms and their susceptibility to environmental variability and climate change. Contemporary microbial oceanography is truly a sea of opportunity and excitement.     

SCOWLP classification: Structural comparison and analysis of protein binding regions

Background  

Detailed information about protein interactions is critical for our understanding of the principles governing protein recognition mechanisms. The structures of many proteins have been experimentally determined in complex with different ligands bound either in the same or different binding regions. Thus, the structural interactome requires the development of tools to classify protein binding regions. A proper classification may provide a general view of the regions that a protein uses to bind others and also facilitate a detailed comparative analysis of the interacting information for specific protein binding regions at atomic level. Such classification might be of potential use for deciphering protein interaction networks, understanding protein function, rational engineering and design.     

Evolution of structurally disordered proteins promotes neostructuralization

Protein structure is generally more conserved than sequence, but for regions that can adopt different structures in different environments, does this hold true? Understanding how structurally disordered regions evolve altered secondary structure element propensities as well as conformational flexibility among paralogs are fundamental questions for our understanding of protein structural evolution. We have investigated the evolutionary dynamics of structural disorder in protein families containing both orthologs and paralogs using phylogenetic tree reconstruction, protein structure disorder prediction, and secondary structure prediction in order to shed light upon these questions. Our results indicate that the extent and location of structurally disordered regions are not universally conserved. As structurally disordered regions often have high conformational flexibility, this is likely to have an effect on how protein structure evolves as spatially altered conformational flexibility can also change the secondary structure propensities for homologous regions in a protein family.     

Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae

Glycosyl-phosphatidylinositol (GPI)-linked proteins are transported to the apical surface of epithelial cells where they undergo cholesterol- dependent clustering in membrane micro-invaginations, termed caveolae or plasmalemmal vesicles. However, the sorting machinery responsible for this caveolar-clustering mechanism remains unknown. Using transfected MDCK cells as a model system, we have identified a complex of cell surface molecules (80, 50, 40, 22-24, and 14 kD) that interact in a pH- and cholesterol-dependent fashion with an apical recombinant GPI-linked protein. A major component of this hetero-oligomeric protein complex is caveolin, a type II transmembrane protein. As this hetero- oligomeric caveolin complex is detectable almost immediately after caveolin synthesis, our results suggest that caveolae may assemble intracellularly during transport to the cell surface. As such, our studies have implications for understanding both the intracellular biogenesis of caveolae and their subsequent interactions with GPI- linked proteins in epithelia and other cell types.