Molecular characterization of the heat-inducible LmSTI1 protein of Leishmania major

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Abstract

We have recently isolated a cDNA encoding the Leishmania major homologue of the yeast stress-inducible protein STI1. Southern blot analyses indicate that this protein is encoded by a single copy gene in L. major and that this gene is highly conserved throughout the Leishmania genus. The STI1 gene is constitutively expressed in both L. major promastigotes and amastigotes however, STI1 transcript levels can be upregulated in promastigotes by a shift in culture temperature from 26 to 37°C. Upregulation of transcript was detectable within 5′ of heat shock and continued to increase for a further 8 h before returning to constitutive levels. In addition, biosynthetic incorporation of [35S]methionine followed by immunoprecipitation revealed an increase in the level of nascent STI1 protein synthesized when promastigote cultures were shifted from 26 to 37°C. The L. major STI1 protein and the heat shock proteins Hsp83 and Hsp70 form a salt-sensitive complex in L. major promastigotes as evidenced by co-immunoprecipitation using an antiserum specific for L. major STI1. Furthermore, this complex can be reconstituted in vitro by adding recombinant STI1 containing an amino-terminal histidine tag to promastigote lysate and subsequent purification using metal chelate affinity chromatography.

Introduction

Heat shock proteins have been characterized as antigenic molecules in a variety of bacterial and parasitic infectious organisms including Mycobacteria [1], Chlamydia [2], Borrelia [3], Plasmodium [4], Leishmania [5]and Trypanosoma [6]. Recently, we described the molecular cloning of a novel protein antigen from Leishmania major (LmSTI1) that was highly similar to a 60 kDa stress-inducible protein (STI1) from Saccharomyces cerevisiae [7]. Yeast STI1 was originally isolated during a genetic screen for proteins that mediate the heat shock response via trans-activation of the SSA4 (Hsp70) promoter [8]. Subsequently, STI1 was found to be a major component of the Hsp90 chaperone complex and has been implicated in the Hsp90-dependent maturation of steroid receptors and the oncogenic tyrosine kinase, pp60v-src 9, 10, 11. STI1, along with several other components of the Hsp90 complex (p23, CyP40, FKBP52 and FKBP54) have thus been classified as Hsp90-associated proteins (Hsp90APs) or `co-chaperones' [10]. A general characteristic of the `co-chaperone' proteins is the presence of a degenerate 34 amino acid motif referred to as the tetratricopeptide (TPR) repeat motif 12, 13. The TPR motif is found in a large and functionally diverse group of proteins and is speculated to mediate protein–protein interactions. The STI1 protein contains six copies of the TPR motif dispersed throughout the complete length of the protein. In S. cerevisiae null mutations in either of STI1 or HSC82 (the constitutively expressed Hsp90 of S. cerevisiae) result in no loss in viability whereas simultaneous mutation of both genes results in severely reduced viability [10]. Furthermore, distinct allele-specific growth effects were observed when STI1 was overexpressed in a panel of strains harboring temperature-sensitive point mutations in HSP82 (the temperature inducible Hsp90 of S. cerevisiae). Together these results suggest that STI1 and Hsp90 interact in vivo and that this interaction is critical for normal cell viability.

STI1 homologues have also been described in humans [14], mouse [15], chicken oviduct [16], rabbit reticulocyte lysate [17]and in soybean [18]and the extent of conservation between this group of proteins is striking considering the evolutionary distance separating them. In addition to forming a complex with Hsp90, the vertebrate STI1 homologue (also known as p60, extendin, IEF SSP 3521, Hsp70 recycling factor (RF-Hsp70) or Hsp70/Hsp90 organizing protein (Hop)) has been shown to act as an ATP/ADP-exchange factor during the recycling of Hsp70 [17]. Also, it is has been demonstrated that STI1 mediates the formation of a complex between Hsp70 and Hsp90 15, 16.

Interestingly, STI1 genes themselves have been shown to be inducible by heat shock or by treatment with the amino acid analogue canavanine 8, 15, 18and the human homologue was identified as a gene that is upregulated in SV40-transformed fibroblasts [14]. For these reasons the STI1 genes have been categorized as `stress-inducible', however, there is no relationship with the `classical' heat shock genes at either the nucleotide or amino acid sequence level. In this report we demonstrate that expression of LmSTI1 in Leishmania promastigotes is induced, at both the RNA and protein levels, by a shift in culture temperature from 26 to 37°C, consistent with LmSTI1 being a member of the `stress-inducible' family of proteins. In addition we demonstrate that, as in other eukaryotes, LmSTI1 forms a salt-sensitive complex with Hsp70 and with Hsp83, the Leishmania Hsp90 homologue.

Section snippets

Southern/Northern blot hybridization analysis

A Southern blot containing genomic DNA (2.5 μg/lane) isolated from L. major, L. tropica, L. donovani, L. infantum, L. chagasi, L. amazonensis, L. braziliensis, L. guyanensis, T. cruzi and T. brucei digested with the restriction enzymes indicated was kindly provided by D. Dillon (this institute). The blot was hybridized with probes generated from the previously described cDNA clone pfl1-1 [7]; Probe A, a BamHI/KpnI restriction fragment that encompasses the complete protein-coding region of the

Evolutionary conservation of STI1 protein sequences

The degree of relatedness between LmSTI1 and other members of the STI1 protein family is apparent in the alignment shown in Fig. 1A. All members of the STI1 protein family contain six dispersed motifs that conform to the TPR consensus motif [12]. An alignment of the six putative TPR motifs of the L. major STI1 protein with the TPR consensus motif is shown in Fig. 1B. Although the TPR domain is found in a functionally diverse array of proteins, the number and arrangement of TPR motifs is protein

Discussion

In this investigation, we have demonstrated that, in addition to being a dominant antigen during experimental murine leishmaniasis [7], the LmSTI1 protein of L. major is expressed in a temperature-dependent manner reminiscent of the classical heat shock proteins. The relationship between these two characteristics is not clear at this time. However, it has been previously observed that heat shock proteins are targeted as antigenic molecules during many protozoan and bacterial infections (for

Acknowledgements

We wish to thank Jeffrey Guderian and Charcha Jen for technical assistance. This work is supported by Grant AI25038 from the National Institutes of Health. John R. Webb is a Fellow of the Medical Research Council of Canada.

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