Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

液泡ATP酶在肿瘤中的作用及机制

液泡ATP酶（vacuolar ATPases,V-ATPases）是真核细胞中高度保守的一类大型多亚基复合物,广泛分布于质膜及溶酶体、内体、囊泡等细胞内膜系统,能够借助水解ATP产生的能量控制H+的跨膜转运。V-ATPases通过对细胞内外多种结构的酸化作用调控着一系列重要的细胞活动,如膜运输、蛋白质加工和降解等。近年来,V-ATPases在肿瘤形成中的功能正逐渐成为研究热点。本文重点综述了V-ATPases通过调控细胞内外环境pH,从而在肿瘤发生过程中所行使的多种功能,例如V-ATPases抑制肿瘤细胞凋亡,参与肿瘤细胞自噬,促进肿瘤的侵袭、迁移与增殖,以及参与肿瘤耐药性的产生等。阐明V-ATPases在肿瘤中的作用机制有望为肿瘤治疗策略的探索、新型药物的开发以及相关科学研究的开展提供参考。     

Structural conservation and functional diversity of V-ATPases

The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H⁺-ATPase that was named V-ATPase. The structure and function of V-ATPases from various sources was extensively studied in the last few years. Several genes encoding subunits of the enzyme were cloned and sequenced. The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes. The two families of proton pumps share structural and functional similarity. They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors. Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium. The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase. They also shed light on the functional assembly of the enzyme in the vacuolar system.     

Ribonuclease P structure and function inArchaea

An important approach to understanding RNA-based catalytic function by ribonuclease P is the investigation of its evolutionary diversity in structure and function. Because RNase P enzymes from all organisms are thought to share common ancestry, the fundamental features of structure and biochemistry should be conserved in all of its modern forms. In contrast to the bacterial enzyme, the RNase P enzymes fromEucarya, organelles, andArchaea are poorly understood. This review describes our nascent understanding of the structure and function of RNase P inArchaea, and how this enzyme compares to its homologs in the other evolutionary Domains.Abbreviations RNase P ribonuclease P - tRNA transfer RNA - pre-tRNA 5-unprocessed precursor transfer RNA - Archaea a.k.a. archaebacteria - Bacteria a.k.a. eubacteria - Eucarya a.k.a. eukaryotic nucleus/cytoplasm     

Structure and Function of the A1A0-ATPases from Methanogenic Archaea

Recent molecular studies revealed nine to ten gene products involved infunction/assembly of the methanoarchaeal ATPase and unravel a closerelationship of the A₁A₀-ATPase and theV₁V₀-ATPase with respect to subunit composition and thestructure of individual subunits. Most interestingly, there is anastonishing variability in the size of the proteolipids in methanoar chaealA₁A₀-ATPases with six, four, or two transmembranehelices and a variable number of conserved protonizable groups per monomer.Despite the structural similarities the A₁A₀-ATPasediffers fundamentally from the V₁V₀-ATPase by itsability to synthesize ATP, a feature shared withF₁F₀-ATPases. The discovery of duplicated andtriplicated versions of the proteolipid in A₁A₀-ATPsynthases questions older views of the structural requirements for ATPsynthases versus ATP hydrolases and sheds new light on the evolutionof these secondary energy converters.     

Evolution of networks and sequences in eukaryotic cell cycle control

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.     

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.     

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.     

Bioenergetics in human evolution and disease: implications for the origins of biological complexity and the missing genetic variation of common diseases

Two major inconsistencies exist in the current neo-Darwinian evolutionary theory that random chromosomal mutations acted on by natural selection generate new species. First, natural selection does not require the evolution of ever increasing complexity, yet this is the hallmark of biology. Second, human chromosomal DNA sequence variation is predominantly either neutral or deleterious and is insufficient to provide the variation required for speciation or for predilection to common diseases. Complexity is explained by the continuous flow of energy through the biosphere that drives the accumulation of nucleic acids and information. Information then encodes complex forms. In animals, energy flow is primarily mediated by mitochondria whose maternally inherited mitochondrial DNA (mtDNA) codes for key genes for energy metabolism. In mammals, the mtDNA has a very high mutation rate, but the deleterious mutations are removed by an ovarian selection system. Hence, new mutations that subtly alter energy metabolism are continuously introduced into the species, permitting adaptation to regional differences in energy environments. Therefore, the most phenotypically significant gene variants arise in the mtDNA, are regional, and permit animals to occupy peripheral energy environments where rarer nuclear DNA (nDNA) variants can accumulate, leading to speciation. The neutralist–selectionist debate is then a consequence of mammals having two different evolutionary strategies: a fast mtDNA strategy for intra-specific radiation and a slow nDNA strategy for speciation. Furthermore, the missing genetic variation for common human diseases is primarily mtDNA variation plus regional nDNA variants, both of which have been missed by large, inter-population association studies.     

Evolution of viral structure

Viruses vastly outnumber their host cells and must present a huge selective pressure. It is also becoming evident that only a small percent of the eukaryotic genome codes for molecules involved in cellular structures and functions, and that much of the remainder may have a viral origin. Viruses clearly play a central role in the biosphere, but how is this viral world organized? Classification was originally based on virus morphology and the particular host infected, but now there is an increasing trend to rely on sequence information. The type of genome (e.g., RNA or DNA, single- or double-stranded) provides fundamental classification criteria, while sequence comparisons can provide fine mapping for closely related viruses. However, it is currently very difficult to identify long-range evolutionary relationships. We present here a different approach, based on the idea that each virus has an innate "self." When the structures and functions characteristic of this "self" are identified, then they uncover relationships beyond those accessible from sequence information alone. The new approach is illustrated by sketching some possible viral lineages. We propose that urviruses were present before the division of cellular life into its current domains, and that the viral world has lineages that can be traced back to the root of the universal tree of life.     

Analysis of evolution of exon-intron structure of eukaryotic genes

The availability of multiple, complete eukaryotic genome sequences allows one to address many fundamental evolutionary questions on genome scale. One such important, long-standing problem is evolution of exon-intron structure of eukaryotic genes. Analysis of orthologous genes from completely sequenced genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists. The data on shared and lineage-specific intron positions were used as the starting point for evolutionary reconstruction with parsimony and maximum-likelihood approaches. Parsimony methods produce reconstructions with intron-rich ancestors but also infer lineage-specific, in many cases, high levels of intron loss and gain. Different probabilistic models gave opposite results, apparently depending on model parameters and assumptions, from domination of intron loss, with extremely intron-rich ancestors, to dramatic excess of gains, to the point of denying any true conservation of intron positions among deep eukaryotic lineages. Development of models with adequate, realistic parameters and assumptions seems to be crucial for obtaining more definitive estimates of intron gain and loss in different eukaryotic lineages. Many shared intron positions were detected in ancestral eukaryotic paralogues which evolved by duplication prior to the divergence of extant eukaryotic lineages. These findings indicate that numerous introns were present in eukaryotic genes already at the earliest stages of evolution of eukaryotes and are compatible with the hypothesis that the original, catastrophic intron invasion accompanied the emergence of the eukaryotic cells. Comparison of various features of old and younger introns starts shedding light on probable mechanisms of intron insertion, indicating that propagation of old introns is unlikely to be a major mechanism for origin of new ones. The existence and structure of ancestral protosplice sites were addressed by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and, accordingly, are not subject to selection for splicing efficiency. It was shown that introns indeed predominantly insert into or are fixed in specific protosplice sites which have the consensus sequence (A/C)AG|Gt.     

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.     

Evolution of avian eggshell structure

Data are presented suggesting that birds have evolved eggs with shells containing different structures (numbers of mammillae per unit of inner eggshell surface area, i.e., mammillary densities) to cope up with different calcium requirements imposed by different growth rates and modes of development. Precocial bird species grow slowly, but have high mammillary density, while altricial bird species grow rapidly, but have low mammillary density. These results suggest an adaptation associated with growth rate and mode of development and show, moreover, that the mammillary layer is indicative of the breeding biology of the bird. J. Morphol. 2012. © 2011 Wiley Periodicals, Inc.     

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder. We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.     

Domain organization and function of Salmonella FliK, a flagellar hook-length control protein

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.     

真核细胞的中间纤维结构、组装和功能的研究

在真核细胞中普遍存在中间纤维(IF),不同类细胞的中间纤维都有相似的“头部 1A L1 1B L1-2 2A L2 2B 尾部”结构,其中：头部有一个β—折叠区,1A上有七个残基的亚结构,1B和2A L2 2B上有规则的轴向排列的氨基酸残基,2B的铲螺旋C末端有一个高度保守的氨基酸残基框,这些结构都有其独特的功能。各种IF组装方式不同,但至少都经历了非纤维性颗粒、波形短纤维和长纤维的形态变化过程。研究发现,IF具有阻止细胞凋亡的功能;在细胞凋亡过程中IF发生磷酸化和降解;细胞质中间纤维(CIF)在染色质一核纤层／CIF结合中发挥DNA选择功能。     

Apoptosis in Unicellular Organisms: Mechanisms and Evolution

Data about the programmed death (apoptosis) in unicellular organisms, from bacteria to ciliates, are discussed. Firstly apoptosis appeared in lower eukaryotes, but its mechanisms in these organisms are different from the classical apoptosis. During evolution, the apoptotic process has been improving gradually, with reactive oxygen species and Ca2+ playing an essential role in triggering apoptosis. All eukaryotic organisms have apoptosis inhibitors, which might be introduced by viruses. In the course of evolution, caspases and apoptosis-inducing factor appeared before other apoptotic proteins, with so-called death receptors being the last among them. The functional analogs of eukaryotic apoptotic proteins take parts in the programmed death of bacteria.     

Sorting of early and late flagellar subunits after docking at the membrane ATPase of the type III export pathway

The bacterial flagellum assembles in a strict order, with structural subunits delivered to the growing flagellum by a type III export pathway. Early rod-and-hook subunits are exported before completion of the hook, at which point a subunit-specificity switch allows export of late filament subunits. This implies that in bacteria with multiple flagella at different stages of assembly, each export pathway can discriminate and sort unchaperoned early and chaperoned late subunits. To establish whether subunit sorting is distinct from subunit transition from the cytosol to the membrane, in particular docking at the membrane-associated FliI ATPase, the pathway was manipulated in vivo. When ATP hydrolysis by the FliI ATPase was disabled and when the pathway was locked into an early export state, both unchaperoned early and chaperoned late subunits stalled and accumulated at the inner membrane. Furthermore, a chaperone that attenuates late subunit export by stalling when docked at the wild-type ATPase also stalled at the ATPase in an early-locked pathway and inhibited export of early subunits in both native and early-locked pathways. These data indicate that the pathways for early and late subunits converge at the FliI ATPase, independent of ATP hydrolysis, before a distinct, separable sorting step. To ascertain the likely signals for sorting, the export of recombinant subunits was assayed. Late filament subunits unable to bind their chaperones were still sorted accurately, but chaperoned late subunits were directed through an early-locked pathway when fused to early subunit N-terminal export signal regions. Furthermore, while an early subunit signal directed export of a heterologous type III export substrate through both native and early-locked pathways, a late subunit signal only directed export via native pathways. These data suggest that subunits are distinguished not by late chaperones but by N-terminal export signals of the subunits themselves.