Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers

Background

The rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins. They are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.

Results

Phylogenetic tree analysis carried out using several independent methods for tree constructions and the corresponding statistical tests suggests that, despite its broad distribution in all three superkingdoms, the rhomboid family was not present in the last universal common ancestor of extant life forms. Instead, we propose that rhomboids evolved in bacteria and have been acquired by archaea and eukaryotes through several independent horizontal gene transfers. In eukaryotes, two distinct, ancient acquisitions apparently gave rise to the two major subfamilies, typified by rhomboid and PARL (presenilins-associated rhomboid-like protein), respectively. Subsequent evolution of the rhomboid family in eukaryotes proceeded by multiple duplications and functional diversification through the addition of extra transmembrane helices and other domains in different orientations relative to the conserved core that harbors the protease activity.

Conclusions

Although the near-universal presence of the rhomboid family in bacteria, archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor, phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer. This emphasizes the importance of explicit phylogenetic analysis for the reconstruction of ancestral life forms. A hypothetical scenario for the origin of intracellular membrane proteases from membrane transporters is proposed.

     

Concerted gene recruitment in early plant evolution

Background  

Horizontal gene transfer occurs frequently in prokaryotes and unicellular eukaryotes. Anciently acquired genes, if retained among descendants, might significantly affect the long-term evolution of the recipient lineage. However, no systematic studies on the scope of anciently acquired genes and their impact on macroevolution are currently available in eukaryotes.     

RNase MRP and the RNA processing cascade in the eukaryotic ancestor

Background

Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor.

Results

We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution.

Conclusion

We conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

     

Evolutionary history and functional implications of protein domains and their combinations in eukaryotes

Background  

In higher multicellular eukaryotes, complex protein domain combinations contribute to various cellular functions such as regulation of intercellular or intracellular signaling and interactions. To elucidate the characteristics and evolutionary mechanisms that underlie such domain combinations, it is essential to examine the different types of domains and their combinations among different groups of eukaryotes.     

A genomic timescale for the origin of eukaryotes

Background  

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.     

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

Background

Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.

Results

We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.

Conclusions

The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.     

Analysis of the role of PCNA-DNA contacts during clamp loading

Background  

Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.     

The iron-sulfur cluster assembly genes <Emphasis Type="Italic">iscS</Emphasis> and <Emphasis Type="Italic">iscU</Emphasis> of <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> were acquired by horizontal gene transfer

Background  

Iron-sulfur (FeS) proteins are present in all living organisms and play important roles in electron transport and metalloenzyme catalysis. The maturation of FeS proteins in eukaryotes is an essential function of mitochondria, but little is known about this process in amitochondriate eukaryotes. Here we report on the identification and analysis of two genes encoding critical FeS cluster (Isc) biosynthetic proteins from the amitochondriate human pathogen Entamoeba histolytica.     

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

Background

Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal Findings

We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions

Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.     

Glutathione analogs in prokaryotes

Background

Oxygen is both essential and toxic to all forms of aerobic life and the chemical versatility and reactivity of thiols play a key role in both aspects. Cysteine thiol groups have key catalytic functions in enzymes but are readily damaged by reactive oxygen species (ROS). Low-molecular-weight thiols provide protective buffers against the hazards of ROS toxicity. Glutathione is the small protective thiol in nearly all eukaryotes but in prokaryotes the situation is far more complex.

Scope of review

This review provides an introduction to the diversity of low-molecular-weight thiol protective systems in bacteria. The topics covered include the limitations of cysteine as a protector, the multiple origins and distribution of glutathione biosynthesis, mycothiol biosynthesis and function in Actinobacteria, recent discoveries involving bacillithiol found in Firmicutes, new insights on the biosynthesis and distribution of ergothioneine, and the potential protective roles played by coenzyme A and other thiols.

Major conclusions

Bacteria have evolved a diverse collection of low-molecular-weight protective thiols to deal with oxygen toxicity and environmental challenges. Our understanding of how many of these thiols are produced and utilized is still at an early stage.

General significance

Extensive diversity existed among prokaryotes prior to evolution of the cyanobacteria and the development of an oxidizing atmosphere. Bacteria that managed to adapt to life under oxygen evolved, or acquired, the ability to produce a variety of small thiols for protection against the hazards of aerobic metabolism. Many pathogenic prokaryotes depend upon novel thiol protection systems that may provide targets for new antibacterial agents. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Horizontal transfer of a eukaryotic plastid-targeted protein gene to cyanobacteria

Background  

Horizontal or lateral transfer of genetic material between distantly related prokaryotes has been shown to play a major role in the evolution of bacterial and archaeal genomes, but exchange of genes between prokaryotes and eukaryotes is not as well understood. In particular, gene flow from eukaryotes to prokaryotes is rarely documented with strong support, which is unusual since prokaryotic genomes appear to readily accept foreign genes.     

Riboswitches as hormone receptors: hypothetical cytokinin-binding riboswitches in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Riboswitches are mRNA elements that change conformation when bound to small molecules. They are known to be key regulators of biosynthetic pathways in both prokaryotes and eukaryotes.     

Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

Background

Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes.

Results

We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists).

Conclusion

LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

     

Differential gene transfers and gene duplications in primary and secondary endosymbioses

Background  

Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, gro EL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of gro EL homologues.