Tài liệu Báo cáo khóa học: TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei pdf

TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei

Stefan Kunz1

, Thomas Kloeckner2

, Lars-Oliver Essen3,*, Thomas Seebeck1 and Michael Boshart2

1

Institute of Cell Biology, University of Bern, Switzerland; 2

Department of Biology I, University of Munich, Germany; 3

MaxPlanck Institute for Biochemistry, Martinsried, Germany

Cyclic nucleotide specific phosphodiesterases (PDEs) are

important components of all cAMP signalling networks.

In humans, 11 different PDE families have been identified to

date, all of which belong to the class I PDEs. Pharmaco￾logically, they have become of great interest as targets for the

development of drugs for a large variety of clinical condi￾tions. PDEs in parasitic protozoa have not yet been exten￾sively investigated, despite their potential as antiparasitic

drug targets. The current study presents the identification

and characterization of a novel class I PDE from the para￾sitic protozoon Trypanosoma brucei, the causative agent of

human sleeping sickness. This enzyme, TbPDE1, is encoded

by a single-copy gene located on chromosome 10, and it

functionally complements PDE-deficient strains of Sac￾charomyces cerevisiae. Its C-terminal catalytic domain shares

about 30% amino acid identity, including all functionally

important residues, with the catalytic domains of human

PDEs. A fragment of TbPDE1 containing the catalytic

domain could be expressed in active form in Escherichia coli.

The recombinant enzyme is specific for cAMP, but exhibits

a remarkably high Km of > 600 lM for this substrate.

Keywords: African trypanosomes; cAMP signaling; class I

phosphodiesterase; sleeping sickness.

Cyclic AMP is involved in the regulation of numerous

biological functions, such as the control of metabolic

pathways in eubacteria [1], differentiation and virulence in

fungi [2], cell aggregation in Dictyostelium [3], transduction

of gustatory and olfactory signals [4], the control of

rhythmic oscillations in heart and brain [5] and learning

and long-term memory formation [6] in multicellular

organisms. In eukaryotic cells, hydrolysis of cAMP by

cyclic nucleotide specific phosphodiesterases (PDEs) is the

only means of rapidly inactivating the cAMP signal. PDEs

represent a large and divergent group of enzymes, and two

distinct PDE classes have been identified [7,8]. Class I

enzymes include all currently known families of mammalian

PDEs, as well as a number of PDEs from lower euk aryotes,

such as PDE2 from the yeast Saccharomyces cerevisiae [8] or

the product of the regA gene of Dictyostelium discoideum [9].

In mammals, 11 distinct class I PDE families have been

identified, based on DNA sequence analysis and on the

pharmacological profiles of the enzymes [10,11]. At the

amino acid level, family members exhibit > 50% sequence

identity within a conserved catalytic core of about 250

amino acids. Between families, the sequence identity drops

to 30–40% in the same region [12], and no significant

similarity is found outside the catalytic domain.

Considering the importance of the PDEs for signal

transduction, it is not unexpected that mutations in PDE

genes have been recognized as the underlying cause of

several genetic diseases [13–15]. In clinical pharmacology,

the PDEs have also become highly attractive targets for

drug development, and a large number of highly family￾specific inhibitors have been developed. PDE inhibitors are

under exploration, or already in clinical use, for ailments as

diverse as autoimmune diseases, arthritis, asthma, impo￾tency and as anti-inflammatory agents (reviewed in

[16–18]).

In view of the spectacular success of PDE inhibitors as

chemotherapeutics, it is surprising how little effort has been

made so far to explore the PDEs of parasites as potential

targets for antiparasitic drugs. The African trypanosome

Trypanosoma brucei is the protozoon that causes the fatal

human sleeping sickness, as well as Nagana, a devastating

disease of domestic animals in large parts of sub-Saharan

Africa. While many aspects of trypanosome cell biology

have been extensively studied, very little is still known about

cAMP signalling [19–22]. Early workhas shown that the

steady-state concentration of cAMP varies during the life

cycle of the parasite in its mammalian host [23]. Vassella

et al. have provided evidence for a crucial role of cAMP

in triggering population-density induced differentiation of

long-slender to short-stumpy bloodstream forms in culture

[24]. An early study on PDEs demonstrated PDE activity in

cell lysates of the bloodstream form of T. brucei [25].

Recently, a small gene family coding for class I PDEs

(TbPDE2) was identified in T. brucei, and their gene

products were characterized as cAMP-specific PDEs [26–

28]. The current study describes the identification of a novel

class I PDE from T. brucei, TbPDE1. This enzyme bears no

sequence similarity to any of the other class I PDE families

Correspondence to T. Seebeck, Institute of Cell Biology,

Baltzerstrasse 4, CH-3012 Bern, Switzerland.

Fax: + 41 31 631 46 84, Tel.: + 41 31 631 46 49,

E-mail: [email protected]

Abbreviations: PDE, cyclic-nucleotide specific phosphodiesterase;

IBMX, isobutyl-methyl-xanthine; IC50, 50% inhibitory

concentrations.

Note: A web site is available at http://www.izb.unibe.ch

*Present address: Department of Chemistry, Hans Meerwein-Strasse,

Philipps University, D-35032 Marburg, Germany.

(Received 16 October 2003, revised 10 December 2003,

accepted 16 December 2003)

Eur. J. Biochem. 271, 637–647 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2003.03967.x