Selenoprotein synthesis and regulation in Archaea

Background

The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism.

Selenoprotein synthesis: UGA does not end the story

It is well established that the beneficial effects of the trace element selenium are mediated by its major biological product, the amino acid selenocysteine, present in the active site of selenoproteins. These fulfill different functions, as varied as oxidation-reduction of metabolites in bacteria, reduction of reactive oxygen species, control of the redox status of the cell or thyroid hormone maturation. This review will focus on the singularities of the selenocysteine biosynthesis pathway and its unique incorporation mechanism into eukaryal selenoproteins. Selenocysteine biosynthesis from serine is achieved on tRNA(Sec) and requires four proteins. As this amino acid is encoded by an in-frame UGA codon, otherwise signaling termination of translation, ribosomes must be told not to stop at this position in the mRNA. Several molecular partners acting in cis or in trans have been identified, but their knowledge has not enabled yet to firmly establish the molecular events underlying this mechanism. Data suggest that other, so far uncharacterized factors might exist. In this survey, we attempted to compile all the data available in the literature and to describe the latest developments in the field.     

Aminoacyl-tRNA synthesis in archaea: different but not unique

Accurate aminoacyl-tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co-translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non-canonical aminoacyl-tRNA synthesis systems most are also found elsewhere albeit less frequently.     

Selenoprotein synthesis: an expansion of the genetic code.

A number of enzymes employ the unusual amino acid selenocysteine as part of their active site because of its high chemical reactivity. Selenocysteine is incorporated into these proteins co-translationally: biosynthesis occurs on a specific tRNA and insertion into a growing polypeptide is directed by a UGA codon in the mRNA. In E. coli, this requires a specific translation factor. Selenocysteine thus represents a unique expansion of the genetic code.     

Selenoprotein expression and function—Selenoprotein W

Selenoprotein W (SeW) is a small selenoprotein (85 to 88 amino acids) first identified in sheep suffering from selenium deficiency. The levels are highest in muscle, heart (except rodents) spleen and brain. The deduced amino acid sequence has been obtained for mice, rats, monkeys, humans, sheep, pigs, fish and chickens. The sequences of SeW are identical in rats and mice as well as monkeys and humans. In all eight species of animals cysteine is present at residue number 9 and selenocysteine at residue number 13. Residue number 37 is cysteine in six species of animal with fish and chickens as the exceptions. Of those examined, the rodent SeW is the only one containing four cysteines whereas the others contain only two cysteines. Glutathionylaltion has been shown for SeW from rats and monkeys but has not been confirmed for this selenoprotein from the other six animals. The biological function of SeW has not been definitely identified. Evidence has been obtained that it can serve as an antioxidant, responds to stress, involved in cell immunity, specific target for methylmercury, and has thioredoxin-like function.     

In vivo requirement of selenophosphate for selenoprotein synthesis in archaea

Biosynthesis of selenocysteine, the 21st proteinogenic amino acid, occurs bound to a dedicated tRNA in all three domains of life, Bacteria, Eukarya and Archaea, but differences exist between the mechanism employed by bacteria and eukaryotes/archaea. The role of selenophosphate and the enzyme providing it, selenophosphate synthetase, in archaeal selenoprotein synthesis was addressed by mutational analysis. Surprisingly, MMP0904, encoding a homologue of eukaryal selenophosphate synthetase in Methanococcus maripaludis S2, could not be deleted unless selD , encoding selenophosphate synthetase of Escherichia coli , was present in trans , demonstrating that the factor is essential for the organism. In contrast, the homologous gene of M. maripaludis JJ could be readily deleted, obviating the strain's ability to synthesize selenoproteins. Complementing with selD restored selenoprotein synthesis, demonstrating that the deleted gene encodes selenophosphate synthetase and that selenophosphate is the in vivo selenium donor for selenoprotein synthesis of this organism. We also showed that this enzyme is a selenoprotein itself and that M. maripaludis contains another, HesB-like selenoprotein previously only predicted from genome analyses. The data highlight the use of genetic methods in archaea for a causal analysis of their physiology and, by comparing two closely related strains of the same species, illustrate the evolution of the selenium-utilizing trait.     

Selenoprotein inAspergillus terreus

Aspergillus terreus, a moderately selenium-tolerant fungus, metabolized⁷⁵ Se-selenite into several protein seleno-amino acids: selenomethionine and selenocysteine, as well as, nonprotein seleno-amino acids, selenocystathionine, and y-glutamyl selenomethyl selenocysteine. The results indicate the failure of the fungus to discriminate between sulphur and selenium. Selenium was also incorporated into several proteins of different molecular weights, mostly of low molecular weight proteins. Labeled studies showed the presence of high levels of selenomethionine and selenocysteine in the protein hydrolysate. The actual incorporation of protein selenoamino acids into the fungal protein was proven. The results demonstrated a finding that detracts from previous held views.     

Bioenergetics of archaea: ATP synthesis under harsh environmental conditions

Archaea are a heterogeneous group of microorganisms that often thrive under harsh environmental conditions such as high temperatures, extreme pHs and high salinity. As other living cells, they use chemiosmotic mechanisms along with substrate level phosphorylation to conserve energy in form of ATP. Because some archaea are rooted close to the origin in the tree of life, these unusual mechanisms are considered to have developed very early in the history of life and, therefore, may represent first energy-conserving mechanisms. A key component in cellular bioenergetics is the ATP synthase. The enzyme from archaea represents a new class of ATPases, the A1A0 ATP synthases. They are composed of two domains that function as a pair of rotary motors connected by a central and peripheral stalk(s). The structure of the chemically-driven motor (A1) was solved by small-angle X-ray scattering in solution, and the structure of the first A1A0 ATP synthases was obtained recently by single particle analyses. These studies revealed novel structural features such as a second peripheral stalk and a collar-like structure. In addition, the membrane-embedded electrically-driven motor (A0) is very different in archaea with sometimes novel, exceptional subunit composition and coupling stoichiometries that may reflect the differences in energy-conserving mechanisms as well as adaptation to temperatures at or above 100 degrees C.     

Recoding in archaea

Standard decoding of the genetic information into polypeptides is performed by one of the most sophisticated cell machineries, the translating ribosome, which, by following the genetic code, ensures the correspondence between the mature mRNA and the protein sequence. However, the expression of a minority of genes requires programmed deviations from the standard decoding rules, globally named recoding. This includes ribosome programmed -/+1 frameshifting, ribosome hopping, and stop codon readthrough. Recoding in Archaea was unequivocally demonstrated only for the translation of the UGA stop codon into the amino acid selenocysteine. However, a new recoding event leading to the 22nd amino acid pyrrolysine and the preliminary reports on a gene regulated by programmed -1 frameshifting have been recently described in Archaea. Therefore, it appears that the study of this phenomenon in Archaea is still at its dawn and that most of the genes whose expression is regulated by recoding are still uncharacterized.     

Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

Cold-adapted archaea

Many archaea are extremophiles. They thrive at high temperatures, at high pressure and in concentrated acidic environments. Nevertheless, the largest proportion and greatest diversity of archaea exist in cold environments. Most of the Earth's biosphere is cold, and archaea represent a significant fraction of the biomass. Although psychrophilic archaea have long been the neglected majority, the study of these microorganisms is beginning to come of age. This review casts a spotlight on the ecology, adaptation biology and unique science that is being realized from studies on cold-adapted archaea.     

RNA-modifying machines in archaea

It has been known for nearly half a century that coding and non-coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non-coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA-containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small-non-coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.     

硒蛋白P的研究进展

微量元素硒 (Ｓｅ)作为许多具有重要生物功能的硒酶的活性中心 ,不但与机体的免疫应答及抗氧化作用等生理功能密切相关 ,而且能够降低癌症的发生率[1,2 ] 。在流行病学和临床研究中 ,常用血浆或全血中Ｓｅ浓度作为衡量Ｓｅ状态的指标 ,而且血浆浓度能比全血浓度更迅速地反映Ｓｅ状态的变化。在哺乳动物血浆中 ,Ｓｅ主要结合在 3种蛋白质中 :硒蛋白Ｐ、胞外谷胱甘肽过氧化物酶和清蛋白。其中硒蛋白Ｐ所含Ｓｅ大约占血浆中全部Ｓｅ浓度的 5 0 %。硒蛋白Ｐ不同于目前所鉴定的所有其他硒蛋白 ,因为它含有 10～ 12个硒代半胱氨酸 (ＳｅＣｙｓ)残…     

Mechanosensitive channels in archaea

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologues in Archaea, the third domain of the phylogenetic tree. Two types of MS channels have been identified in the cell membranes of Haloferax volcanii using the patch clamp technique. Recently MS channels were identified and cloned from two archaeal species occupying different environmental habitats. These studies demonstrate that archaeal MS channels share structural and functional homology with bacterial MS channels. The mechanical force transmitted via the lipid bilayer alone activates all to date known prokaryotic MS channels. This implies the existence of a common gating mechanism for bacterial as well as archaeal MS channels according to the bilayer model. Based on recent evidence that the bilayer model also applies to eukaryotic MS channels, mechanosensory transduction probably originated along with the appearance of the first life forms according to simple biophysical principles. In support of this hypothesis the phylogenetic analysis revealed that prokaryotic MS channels of large and small conductance originated from a common ancestral molecule resembling the bacterial MscL channel protein. Furthemore, bacterial and archaeal MS channels share common structural motifs with eukaryotic channels of diverse function indicating the importance of identified structures to the gating mechanism of this family of channels. The comparative approach used throughout this review should contribute towards understanding of the evolution and molecular basis of mechanosensory transduction in general.     

Selenoprotein P Controls Oxidative Stress in Cornea

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.     

Selenoprotein synthesis in E. coli. Purification and characterisation of the enzyme catalysing selenium activation.

The product of the selD gene from Escherichia coli catalyses the formation of an activated selenium compound which is required for the synthesis of Sec-tRNA (Sec, selenocysteine) from Ser-tRNA and for the formation of the unusual nucleoside 5-methylaminomethyl-2-selenouridine in several tRNA species. selD was overexpressed in a T7 promoter/polymerase system and purified to apparent homogeneity. Purified SELD protein is a monomer of 37 kDa in its native state and catalyses a selenium-dependent ATP-cleavage reaction delivering AMP and releasing the beta-phosphate as orthophosphate. The gamma-phosphate group of ATP was not liberated in a form able to form a complex with molybdate. It was precluded that any putative covalent or non-covalent ligand of SELD not removed during purification participated in the reaction. In a double-labelling experiment employing [75Se]selenite plus dithiothreitol and [gamma-32P]ATP the 75Se and 32P radioactivities co-chromatographed on a poly(ethyleneimine)-cellulose column. No radioactivity originating from ATP eluted in this position when [alpha-32P]ATP or [beta-32P]ATP or [14C]ATP were offered as substrates. The results support the speculation that the product of SELD is a phosphoselenoate with the phosphate moiety derived phosphoselenoate from the gamma-phosphate group of ATP. The alpha,beta cleavage of ATP is also supported by the finding that neither adenosine 5'-[alpha,beta-methylene]triphosphate nor adenosine 5'-[beta,gamma-methylene]triphosphate served as substrates in the reaction.     

Translational recoding in archaea

Translational recoding includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ±1 frameshifting, and ribosome bypassing, which have been found in organisms from all domains of life. They serve to regulate protein expression at translational level and represent a relatively less known exception to the traditional central ‘dogma’ of biology that information flows as DNA→RNA→protein and that it is stored in a co-linear way between the 5′→3′ of nucleic acids and N→C-terminal of polypeptides. In archaea, in which translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, only one case of programmed ?1 frameshifting has been reported so far and further examples, although promising, have not been confirmed yet. We here summarize the current state-of-the-art of this field that, especially in archaea, has relevant implications for the physiology of life in extreme environments and for the origin of life.