Plasmodia express two threonine-peptidase complexes during asexual development

https://doi.org/10.1016/j.molbiopara.2006.03.001Get rights and content

Abstract

Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death.

In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.

Introduction

Malaria remains one of the major health problems worldwide and measures to control the disease in highly endemic regions are currently limited to effective chemotherapy. Genomic and proteomic studies of different plasmodial species and strains promise to aid the development of novel therapeutic strategies to combat parasite infection. The genome of plasmodia is unusual in that it has a very high AT-content and is relatively large in size compared to organisms that contain a similar number of genes, such as Saccharomyces cerevisiae. The large size of the genome correlates with the tendency of plasmodial proteins to be larger than their relatives in other organisms [1]. Increased size, homo- and hetero-polymeric repeats between and within globular domains, the high mutation rate at many protein surfaces due to positive selection by the host's immune system, and strong oscillations in host body-temperature are challenges to the protein folding and degradation apparatus of plasmodia. Accumulation of non-functional or misfolded proteins would otherwise lead to cell death. Protein quality control within living cells is regulated by two main mechanisms: (i) chaperone- and chaperonin-mediated protein folding/re-folding or (ii) protein degradation, should correct folding be unachievable [2]. The two activities can be unified in one enzyme (e.g., bacterial DegP [3]) but in eukaryotes these tasks are performed by distinct protein complexes, whereby the proteasome is responsible for degradation. The proteasome and its prokaryotic predecessor ClpQ/hslV are members of the T1-family of threonine-peptidases which are encoded by two or three genes in archaea, 14 genes in yeast, 16 or 17 genes in vertebrates, and only one gene in bacteria. Both threonine-peptidase complexes self-compartmentalize to form barrel-like structures where the proteolytic activity is located within the central cavity, thereby restricting access to the unspecific proteolytic site. These two proteolytically active ‘core’ particles represent a ∼200 kDa-homododecamer (12 ClpQ/hslV subunits) and a ∼700 kDa-heteroicosikaioctamer (14 α- and 14 β-subunits), respectively. Proteins destined for degradation are actively channeled through associated chaperone complexes, thus providing substrate specificity to the complex.

The genome of P. falciparum contains 14 genes encoding proteasome subunit proteins and, in addition, one gene encoding the ClpQ/hslV ortholog [4]. It is not known whether P. falciparum hslV (subsequently named PfhslV) is expressed and functional and if plasmodial proteasomes are indeed active. However, inhibitor studies strongly suggest an important role of their activity during parasite development [5]. Therefore we investigated primary sequences, expression patterns, complex formation, and enzymatic activities of both protease complexes and evaluated a threonine-peptidase inhibitor for its potential use as a lead compound to develop a new class of antimalarial drugs targeting the plasmodial proteasome and PfhslV.

Section snippets

Sequence analysis

Total RNA was extracted from asynchronously growing P. falciparum cultures and reverse transcribed using superscript III reverse transcriptase (Invitrogen). PfhslV was amplified using a set of primers with overlapping sequence coverage (1: TGGTTTCTCAAGGAACGATG, 2: ATACAATGCTCTTGCAGCTG, 3: CATTTGCATCACACGACATG, 4: GACAACACATATCTGCAGCA, 5: AAACCTAGGCAATGTTTCACAAATAAAATTATT) from cDNA and genomic DNA to confirm the published sequences. The 5′-sequence of PfhslV was analyzed with a 5′/3′ RACE kit

Sequence analysis

Fourteen proteasome subunits and one ClpQ/hslV homolog were identified in the genomic sequence of P. falciparum by blast searches using S. cerevisiae and Escherichia coli sequences as query, respectively. The multiple sequence alignment exhibited a high degree of overall conservation. Coding regions of low complexity (LCRs), typically coding for asparagine, lysine and glutamic acid are a feature of many proteins in plasmodia. Proteasome subunits β1, β3, and β7 contain LCRs (Fig. 1, regions

Discussion

Malaria research was facilitated significantly by the availability of genomic sequences of some plasmodial species and the analysis of whole genome expression profiles during the different stages of plasmodial life. Nevertheless, we are far from an integrated picture of the molecular biology of the parasite, mainly because of the lack of functional data and the difficulty to interfere with regulatory mechanisms of plasmodia. Protein degradation is essential for all living organisms and is,

Acknowledgements

The work received support from the fortüne program (project number: 1195-0-0) of the University of Tübingen.

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    These authors contributed equally to the work.

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