Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCP^O to the red OCP^R form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP’s N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

     

Single Holliday junctions are intermediates of meiotic recombination

Crossing-over between homologous chromosomes facilitates their accurate segregation at the first division of meiosis. Current models for crossing-over invoke an intermediate in which homologs are connected by two crossed-strand structures called Holliday junctions. Such double Holliday junctions are a prominent intermediate in Saccharomyces cerevisiae meiosis, where they form preferentially between homologs rather than between sister chromatids. In sharp contrast, we find that single Holliday junctions are the predominant intermediate in Schizosaccharomyces pombe meiosis. Furthermore, these single Holliday junctions arise preferentially between sister chromatids rather than between homologs. We show that Mus81 is required for Holliday junction resolution, providing further in vivo evidence that the structure-specific endonuclease Mus81-Eme1 is a Holliday junction resolvase. To reconcile these observations, we present a unifying recombination model applicable for both meiosis and mitosis in which single Holliday junctions arise from single- or double-strand breaks, lesions postulated by previous models to initiate recombination.     

Structure of the glucanase inhibitor protein (GIP) family from phytophthora species suggests coevolution with plant endo-beta-1,3-glucanases

During invasion of their plant hosts, species of the oomycete genus Phytophthora secrete glucanase inhibitor proteins (GIPs) into the plant apoplast, which bind and inhibit the activity of plant extracellular endo-beta-1,3-glucanases (EGases). GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH). To study the evolutionary relationship between GIPs and functional SP, database searches were used to identify 48 GIP homologs in the P. sojae, P. ramorum, and P. infestans genomes, composing GIPs, SPH, and potentially functional SP. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Studies of the temporal expression of a four-membered GIP family from P. infestans (PiGIP1 to PiGIP4) further revealed that the genes show distinctly different patterns during an infection timecourse. Codon evolution analyses of GIP homologs identified several positively selected peptide sites and structural modeling revealed them to be in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of a coevolving molecular arms race.     

Hsp90n - An accidental product of a fortuitous chromosomal translocation rather than a regular Hsp90 family member of human proteome

Human cells express two isoforms of the Hsp90 protein, called Hsp90alpha and Hsp90beta. Although existence of the third form called Hsp90alphaDeltaN, or Hsp90N was reported in 1998, our investigation, based on the sequence analysis and attempts to reproduce previous results, demonstrate that there is no evidence that Hsp90N gene is present in human genome and no homologs of such a protein are present in other known eukaryotic genomes. We propose that Hsp90N was created as an artifact of a cDNA synthesis or that it is a chimeric protein, being a result of the chromosomal rearrangement that occurred in a single cell line, after this line was established.     

SPEM: improving multiple sequence alignment with sequence profiles and predicted secondary structures

MOTIVATION: Multiple sequence alignment is an essential part of bioinformatics tools for a genome-scale study of genes and their evolution relations. However, making an accurate alignment between remote homologs is challenging. Here, we develop a method, called SPEM, that aligns multiple sequences using pre-processed sequence profiles and predicted secondary structures for pairwise alignment, consistency-based scoring for refinement of the pairwise alignment and a progressive algorithm for final multiple alignment. RESULTS: The alignment accuracy of SPEM is compared with those of established methods such as ClustalW, T-Coffee, MUSCLE, ProbCons and PRALINE(PSI) in easy (homologs) and hard (remote homologs) benchmarks. Results indicate that the average sum of pairwise alignment scores given by SPEM are 7-15% higher than those of the methods compared in aligning remote homologs (sequence identity <30%). Its accuracy for aligning homologs (sequence identity >30%) is statistically indistinguishable from those of the state-of-the-art techniques such as ProbCons or MUSCLE 6.0. AVAILABILITY: The SPEM server and its executables are available on http://theory.med.buffalo.edu.     

MVB-12, a fourth subunit of metazoan ESCRT-I, functions in receptor downregulation

After ligand binding and endocytosis, cell surface receptors can continue to signal from endosomal compartments until sequestered from the cytoplasm. An important mechanism for receptor downregulation in vivo is via the inward budding of receptors into intralumenal vesicles to form specialized endosomes called multivesicular bodies (MVBs) that subsequently fuse with lysosomes, degrading their cargo. This process requires four heterooligomeric protein complexes collectively termed the ESCRT machinery. In yeast, ESCRT-I is a heterotetrameric complex comprised of three conserved subunits and a fourth subunit for which identifiable metazoan homologs were lacking. Using C. elegans, we identify MVB-12, a fourth metazoan ESCRT-I subunit. Depletion of MVB-12 slows the kinetics of receptor downregulation in vivo, but to a lesser extent than inhibition of other ESCRT-I subunits. Consistent with these findings, targeting of MVB-12 to membranes requires the other ESCRT-I subunits, but MVB-12 is not required to target the remaining ESCRT-I components. Both endogenous and recombinant ESCRT-I are stable complexes with a 1:1:1:1 subunit stoichiometry. MVB-12 has two human homologs that co-localize and co-immunoprecipitate with the ESCRT-I component TSG101. Thus, MVB-12 is a conserved core component of metazoan ESCRT-I that regulates its activity during MVB biogenesis.     

Toward unraveling membrane biogenesis in mammalian autophagy

Autophagy is a unique form of membrane trafficking, which delivers macromolecules and organelles from the cytoplasm to lysosomes for degradation. This fundamental and ubiquitous process in eukaryotic cells is mediated by the double-membrane-bound structures called autophagosomes, which transiently emerge in the cytoplasm. The recent remarkable explosion of knowledge of autophagy has revealed its multiple roles, especially in mammals; in addition to its basic role in turnover and reuse of cellular constituents, the process unexpectedly functions in elimination of invading bacteria and antigen presentation. Analysis of mammalian homologs of the autophagy-related (Atg) proteins identified in yeast has shed light on not only the common molecular mechanisms underlying autophagosome formation, but also specialized mechanisms that are related to the diverse functions and complex regulation of autophagy in higher organisms.     

Calcium-modulated contractile proteins associated with the eucaryotic centrosome

Affinity-purified antibodies that recognize the 20,000-dalton molecular weight (20 kd) striated flagellar root protein of Tetraselmis striata have been used to identify antigenic homologs in other eucaryotic organisms of diverse evolutionary origins. Among the green algae, Tetraselmis and Chlamydomonas, and their colorless relative, Polytomella, the 20-kd homologs appear associated with basal bodies. This occurs most prominently in the form of flagellar roots of both striated and microtubule subtended types. Among cultured mammalian cells (PtK2 and primary mouse macrophage cell lines), flagellar root protein homologs appear as basal feet, pericentriolar fibrils, and pericentriolar satellites. Mammalian sperm cells also show flagellar root protein homologs associated with their basal bodies. We envisage a functional role for these fibrous calcium-sensitive contractile proteins in altering the orientation of centrioles or basal bodies with their associated MTOCs by responding to topological calcium fluxes.     

In-silico analysis of genomic distribution and functional association of hipBA toxin-antitoxin (TA) homologs in entomopathogen Xenorhabdus nematophila

Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila.     

Human cytomegalovirus (HCMV) smallest capsid protein identified as product of short open reading frame located between HCMV UL48 and UL49.

The capsid of cytomegalovirus contains an abundant, low-molecular-weight protein whose coding sequence within the viral genome had not been identified. We have used a combination of biochemical and immunological techniques to demonstrate that this protein, called the smallest capsid protein in human cytomegalovirus, is encoded by a previously unidentified 225-bp open reading frame (ORF) located between ORFs UL48 and UL49. This short ORF, called UL48/49, is the positional homolog of herpes simplex virus ORF UL35 (encoding capsid protein VP26) and shows partial amino acid sequence identity to positional homologs in human herpes viruses 6 and 7.     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

The structure of the minimal relaxase domain of MobA at 2.1 A resolution

The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.     

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.     

Novel insights into the origin and diversification of photosynthesis based on analyses of conserved indels in the core reaction center proteins

The evolution and diversification of different types of photosynthetic reaction centers (RCs) remains an important unresolved problem. We report here novel sequence features of the core proteins from Type I RCs (RC-I) and Type II RCs (RC-II) whose analyses provide important insights into the evolution of the RCs. The sequence alignments of the RC-I core proteins contain two conserved inserts or deletions (indels), a 3 amino acid (aa) indel that is uniquely found in all RC-I homologs from Cyanobacteria (both PsaA and PsaB) and a 1 aa indel that is specifically shared by the Chlorobi and Acidobacteria homologs. Ancestral sequence reconstruction provides evidence that the RC-I core protein from Heliobacteriaceae (PshA), lacking these indels, is most closely related to the ancestral RC-I protein. Thus, the identified 3 aa and 1 aa indels in the RC-I protein sequences must have been deletions, which occurred, respectively, in an ancestor of the modern Cyanobacteria containing a homodimeric form of RC-I and in a common ancestor of the RC-I core protein from Chlorobi and Acidobacteria. We also report a conserved 1 aa indel in the RC-II protein sequences that is commonly shared by all homologs from Cyanobacteria but not found in the homologs from Chloroflexi, Proteobacteria and Gemmatimonadetes. Ancestral sequence reconstruction provides evidence that the RC-II subunits lacking this indel are more similar to the ancestral RC-II protein. The results of flexible structural alignments of the indel-containing region of the RC-II protein with the homologous region in the RC-I core protein, which shares structural similarity with the RC-II homologs, support the view that the 1 aa indel present in the RC-II homologs from Cyanobacteria is a deletion, which was not present in the ancestral form of the RC-II protein. Our analyses of the conserved indels found in the RC-I and RC-II proteins, thus, support the view that the earliest photosynthetic lineages with living descendants likely contained only a single RC (RC-I or RC-II), and the presence of both RC-I and RC-II in a linked state, as found in the modern Cyanobacteria, is a derivation from these earlier phototrophs.     

YidC,a newly defined evolutionarily conserved protein,mediates membrane protein assembly in bacteria

Membranes contain proteins that catalyze a variety of reactions, which lead to the selective permeability of the membrane. For membrane proteins to function as receptors, transporters, channels, and ATPases, they must be targeted to their correct membrane and inserted into the lipid bilayer. Recently, a new membrane component called YidC was discovered that mediates the insertion of proteins into membranes in bacteria. YidC homologs also exist in mitochondria and chloroplasts. Depletion of YidC from the cell interferes with the insertion of membrane proteins that insert both dependent and independent of the SecYEG/SecDFYajC machinery. YidC directly interacts with membrane proteins during the membrane protein insertion process and assists in the folding of the hydrophobic regions into the membrane bilayer. The chloroplast and bacterial YidC homologs are truly functional homologs because the chloroplast homolog Alb3 functionally complements the bacterial YidC depletion strain. The role of YidC in the membrane insertion pathway will be reviewed.     

F-box蛋白COI1稳定性调控机制

拟南芥F-box蛋白COI1(Coronatine insensitive 1)与ASK1(Arabidopsis serine/Threonine kinase 1)蛋白及CUL1(CULLIN1)蛋白等结合形成SCFCOI1泛素连接酶复合体.COI1感知茉莉素信号、进而调控植物一系列的防御反应和生长发育过程.虽然多种作物的COI1同源蛋白已经被相继鉴定,但是其自身蛋白水平的调控机制仍然未知.本文重点研究了蔬菜作物番茄(Solanum lycopersicum)和经济作物烟草(Nicotiana attenuata)中COI1蛋白稳定性的调控机制.结果证明,这两种作物的COI1蛋白通过与ASK1的相互作用而得到稳定,表明形成SCFCOI1复合体可能有助于COI1蛋白的稳定.同时,26S蛋白酶体抑制剂能够明显抑制COI1的降解,说明泛素-蛋白酶体途径参与了其降解过程.这些结果证明在这两个不同物种中,两条互相拮抗的途径共同发挥作用,平衡并稳定COI1蛋白在细胞内的恰当丰度.该研究为深入研究不同作物的茉莉素信号转导调控机制奠定了良好的基础.