*Laboratory for Protozoology, Prince Leopold Institute for Tropical Medicine, Antwerpen, Belgium;
Laboratoire de Chimie Biologique, Université de Mons-Hainaut, Belgium;
Applied Genetics, IBMM, Université Libre de Bruxelles, Gosselies, Belgium
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Abstract |
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Introduction |
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In eukaryotes the 26S proteasome degrades proteins that have been targeted for destruction by the ubiquitin pathway. The catalytic core of the proteasome or 20S particle is an 700-kDa complex of 28 subunits ranging from 20 to 35 kDa in molecular weight and from 4.5 to 8.7 in pI. They show a remarkable structural similarity and extensive sequence homologies at the amino acid level. As viewed under the electron microscope, the 20S complex is a hollowed cylinder made of four stacked rings of seven subunits each. Alpha subunits (
17) form the two outer rings and beta subunits (ß17), the inner two ones. The latter assume the actual proteolytic activity (Coux, Tanaka, and Goldberg 1996
; Bochtler et al. 1999
; Voges, Zwickl, and Baumeister 1999
). In Archea and Actinomycetales one or two representatives of each alpha and beta families are present (Coux, Tanaka, and Goldberg 1996
; De Mot et al. 1999
; Voges, Zwickl, and Baumeister 1999
). On its own the eukaryotic 20S proteasome degrades only small peptides, and it is only when associated with the 19S particle or regulatory complex that it is able to process proteins. The 19S complex contains 17 subunits, including six ATPase subunits forming a hetero-hexameric ring at the base connecting with the 20S proteasome. The 19S complex is believed to recognize polyubiquinated proteins and unfold and translocate them into the 20S particle (Coux, Tanaka, and Goldberg 1996
; Voges, Zwickl, and Baumeister 1999
; Ferrell et al. 2000
). In Archea and Actinomycetales several ATPases that are candidates for activators of their respective proteasomes have been identified (De Mot et al. 1999
; Voges, Zwickl, and Baumeister 1999
).
In eubacteria other than Actinomycetales, ATP-dependent proteolysis is performed by simpler complexes in which ATPase subunits associate with proteolytic ones, i.e., ClpA and ClpX with ClpP, HslU with HslV (Gottesman 1996
; Bochtler et al. 1999
; De Mot et al. 1999
). Although the ClpX and HslU ATPases are both members of the HSP100/Clp family, the HslV protease is not related to serine protease ClpP but rather to ß subunits of the proteasome. Indeed, HslV and proteasome ß subunits share
20% similarity over
200 amino acids, a similar fold and threonine catalytic mechanism (Coux, Tanaka, and Goldberg 1996
; Gottesman 1996
; Bochtler et al. 1999
). Two hexameric (HslV) or heptameric (ClpP) rings of identical proteolytic subunits form the proteolytic complex, which can be capped on either or both ends by a ring made of six identical ATPase subunits. Like the 19S particle in eukaryotes, they are thought to recognize and unfold the protein substrates and translocate them into the proteolytic chamber (Horwich, Weber-ban, and Finley 1999
; Voges, Zwickl, and Baumeister 1999
). Finally, in addition to activating proteolytic complexes, ATPases homopolymeric complexes have a chaperone capability of their own (Wickner, Maurizi, and Gottesman 1999
). So far, the presence of a proteasome or an HslVU complex was considered mutually exclusive (De Mot et al. 1999
). Here, we describe the presence of HslV and HslU subunits coding sequences and their expression in trypanosomatid protozoa. To our knowledge this is the first report of an HslVU complex in a eukaryote and, consequently, the first report of simultaneous occurrence of both a proteasome and an HslVU complex in living organisms.
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Materials and Methods |
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Computer-Assisted Analysis of Sequences and Database Searches
Most nucleotide and amino acid sequence analyses as well as searches in GENBANK and EMBL databases were performed using programs from the Genetic Computer Group (GCG) suite version 10. Prediction of leader peptides and subcellular localizations were performed with the SignalP 1.1 (Nielsen et al. 1997
) and TargetP 1.0 (Emmanuelson et al. 2000
) programs, respectively, at the Danish Center for Biological Sequence Analysis (CBS) server (http://www.cbs.dtu.dk/services/). Keyword interrogations of the expressed sequences tags database (DBEST) and of the genome database (entrez: genome) were performed at the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov). Finally, genome survey sequences (gss) from the Trypanosoma brucei genome project were interrogated by TBLASTN, using the prototypical E. coli HslV (accession number P31059) and HslU (accession number P32168) amino acid sequences as queries. The first hits obtained with the E. coli queries were used in turn to interrogate the databases.
Sequence Alignment and Phylogenetic Analysis
Amino acid sequences were aligned using the Clustal program (Thompson et al. 1997
) as implemented in the Clustal X version 1.8 package (http://www-igbmc.u-strasbg.fr/BioInfo/). Alignments were visualized and edited using the Genedoc package version 2.6 (http://www.psc.edu/biomed/genedoc/). Phylogenetic analysis by the distance method, using neighbor-joining for the construction of dendrograms, was performed using programs in the Clustal package. The options "exclude positions with gaps" and "correct for multiple substitutions" were invoked as indicated in the text. Bootstrap values were calculated on 1,000 repeats of the initial data set. The analysis was repeated using programs from the Phylip package version 3.5c (http://evolution.genetics.washington.edu/phylip.html). One thousand repeats of the Clustal alignment were obtained by SEQBOOT. Distances were computed by PROTDIST, using Dayhoff PAM matrix. Nonrooted trees were constructed by neighbor-joining with NEIGHBOR and a consensus tree was obtained with CONSENSE.
For the study of HslV, representative proteasome ß subunit amino acid sequences from Arabidopsis thaliana, Homo sapiens, and Saccharomyces cerevisiae as well as available ones from the trypanosomatids Leishmania and Trypanosoma were retrieved from public databases and aligned with Clustal. Profile alignment was used to align available HslVs to proteasome ß subunits. First, an unrooted distance dendrogram was constructed by neighbor-joining from the alignment of the mature proteins only (i.e., omitting propeptides). Next, phylogenetic analysis was performed on the HslVs aligned with either the archetypal ß subunit of Thermoplasma acidophilum (acc. P28061), or subunit ß5 from T. brucei (acc. AJ132959) or S. cerevisiae (acc. P30656) as an out-group.
Similarly, available eubacterial HslU sequences were retrieved from the databases and aligned. Selected members of the ClpX were included too. Alignments of the entire proteins or only regions homologous to the N-terminal domain and the C-terminal domain of HslU were used for phylogenetic analysis.
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Results |
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HslV Homologs Are Also Expressed in the Related Trypanosomatids T. (Trypanozoon) brucei and T. (Schizotrypanum) cruzi
A search in the database of expressed sequence tags (DBEST, National Center for Biotechnology Information) pointed to possible bacterial HslV in T. (S.) cruzi epimastigotes (accession numbers AA952689, AA890822, AA952660) and T. (T.) brucei rhodesiense bloodstream forms (AA689168). The sequences were retrieved from the databases and analyzed. Moreover, one of the T. (S.) cruzi clones (clone TENS 1873) was kindly provided by the authors and resequenced (AJ298868). The typical spliced leader sequence was observed at the 5' end of the T. (S.) cruzi cDNA, confirming the kinetoplastic origin of the material. Both T. (S.) cruzi and T. (T.) brucei rhodesiense partial calculated proteins presented high similarity with eubacterial HslVs (table 1
), absence of the ß subunit proteasome signature, possible cleavage site yielding a N-terminal threonine, and conservation of residues Asp17 and Lys33 in the mature protein (fig. 1a
). Mitochondrial localization was suggested by the same arguments as described above (table 1
). The observations thus indicated the presence of HslV homologs in members of the genus Trypanosoma and their transcription into mature messengers, at least in the developmental stages used for constructing the cDNA libraries.
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The properties of the coding sequences and calculated HslUs from L. major, L. infantum, and T. brucei are presented in table 1 and figure 3
. Features supporting their identification with genuine HslU homologs are 55-kDa molecular weight, presence of an ATP-GTP binding site (P-loop, Prosite P00017) near the N-terminus (positions 101118 in fig. 3
), and
60% similarity to the prototypical HslU of E. coli. Furthermore, residues found to be essential to activities of the HslVU complex in mutagenesis studies of E. coli HslU (Song et al. 2000
) were conserved. They were Arg325, Arg393, Glu321, Lys80, and Glu286. Residue Tyr91, which is central to the translocation pore (residues 8795) and a key element in the model of Wang et al. (2001)
, was replaced by similar Phe91 as in the related FtsH protein (Wang et al. 2001
) and in the Helicobacter pylori sequence (fig. 3
). Throughout the bacterial and trypanosomatid sequences, high similarity was observed in regions corresponding to the highly structured N-terminal (N, residues 2109 and 244332 on the E. coli sequence) and C-terminal (C, residues 333443) domains as defined by Bochtler et al. (2000)
from crystal structures of HslU in E. coli. On the contrary, the intermediate domain (I, residues 110243) was less conserved in bacteria and trypanosomatids alike and had several insertions and deletions. In mutagenesis studies the intermediate region supports some degree of deletions and insertions without loss of activity (Song et al. 2000
).
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Discussion |
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In parasitic protozoa, including L. mexicana (Robertson 1999
), T. cruzi (Gonzalez et al. 1996
), and T. brucei (Hua et al. 1996
), a typical eukaryotic proteasome has been biochemically purified, and its role has been explored. Trypanosomatids would therefore be endowed with both a cytoplasmic-eukaryotic and a mitochondrial-eubacterial machinery for degradation of proteins. This is reminiscent of the compartmentalization of enzymes of the glycolytic pathway, where cytoplasmic and glycosomal isoforms of glyceraldehyde-3-phosphate dehydrogenase glycosomal (GAPDH) are coded for by different genes and have a different evolutionary origin. Glycosomal GAPDH is present in the common ancestor to kinetoplastids, whereas the cytoplasmic isoform was acquired at a later stage by horizontal transfer (Hannaert, Opperdoes, and Michels 1998
).
In E. coli, HslV and HslU are heat shock proteins involved in the proteolysis of misfolded proteins (Missiakas et al. 1996
). HslVU and the HslU homopolymeric complexes are also implicated in the regulation of cell division in E. coli through regulation of cell division inhibitor SulA (Kanemori, Yanagi, and Yura 1999
; Seong et al. 1999
, 2000
).
In eukaryotes, homologs of other bacterial ATP-dependent proteases such as Lon and ClpXP (but not HslVU so far) have been found in the mitochondria. They seem essential for biogenesis and maintenance of the mitochondria through degradation of nonassembled or misfolded polypeptides and control of the steady-state levels of regulatory proteins (Käser and Langer 2000
). Similarly, HslVU might complement or supplement other complexes in protein turnover of the mitochondrion in trypanosomatids. It may also have specific functions related to the peculiar biology of digenetic parasites which undergo dramatic morphological and, at least for T. brucei, metabolic remodeling (Vickerman 1994
), including the mitochondrion, as they change host as well as differentiate from one stage to the next in the life cycle.
The observation of eubacterial genes in eukaryotic nuclear genomes is usually seen as evidence of migration of genes from the early endosymbiotic bacteria at the origin of the mitochondria or even evidence of ancient genome fusion at the very origin of the eukaryotic lineage (Gray, Burger, and Lang 1999
). Accordingly, kinetoplastid HslV and HslU might have been present in early eukaryotes and retained in the kinetoplastids but might have been lost in more recent lineages. Indeed, kinetoplastids have been assigned a primordial status and in numerous phylogenetic reconstructions they branch earlier than most lineages of eukaryotes (Vickerman 1994
; Stevens et al. 2001
). Alternatively, a horizontal transfer event in the last common ancestor of trypanosomatids would account for the presence of the gene in the three species. These organisms have ample opportunities of contact with bacteria in the gut of insect vectors. This is best illustrated by the observation of an endosymbiotic ß-proteobacteria in some species of monogenic genera Crithidia, Blastocrithidia, and Herpetomonas (de Souza and Machado Motta 1999
). A search for HslV and HslU homologs in free-living kinetoplastids or in related primitive eukaryotes such as the euglenids could help resolve these questions.
Our phylogenetic analyses indicated that trypanosomatid HslV and HslU, despite clustering with eubacterial sequences, could not be confidently assigned to any particular bacterial clade. Although there is now a large scientific support for the notion that mitochondria evolved once from an endosymbiotic a-proteobacterium, only 200 out of
400 proteins in the yeast mitochondrial proteome can be related to bacteria, and only
50 of them can be closely related to
-proteobacteria (Kurland and Anderson 2000
). Therefore, the lack of safe clustering with the a-proteobacteria sequences does not exclude early mitochondrial origin for trypanosomatid HslV and HslU.
In summary, we present evidence that the eubacterial HslVU and the eukaryotic multicatalytic protease are simultaneously present in trypanosomatid protozoa and expressed, at least in the developmental stages studied. We believe that exploring the role of HslVU in trypanosomatid protozoa could further our knowledge of the metabolic and developmental processes in these important human pathogens. A search for HslV and HslU homologs in free-living kinetoplastids and in other early-branching eukaryotes would help improve our understanding of aspects of mitochondria origins and eukaryotes evolution.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keywords: trypanosomatids
prokaryotes
ATP-dependent protease
evolution
Address for correspondence and reprints: Bernard Couvreur, Virology Department, Veterinary and Agrochemical Research Centre (VAR), Groeselenberg 99, B-1180 Bruxelles, Belgium. E-mail: becou{at}var.fgov.be
.
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