Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Abstract

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems.

The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions

“…Several other reports have analyzed the effects of overexpression of the J domains of cognate or non-cognate JDPs in related/divergent model systems (13,62,63), but rarely has the specific impact of the JDP substrate-binding domain been investigated. Our data, together with those of others, reveal novel insights into the biology of DnaJ family proteins.…”

The heat shock protein 40 family of the malaria parasite Plasmodium falciparum

“…PFD0462w (PfJ1, PfDnaJA) has been shown to display increased mRNA and protein levels upon heat shock (16,17). Heterologous complementation studies have revealed that replacing the bacterial J-domain of a thermosensitive E. coli strain (OD259) with the J-domain from PFD0462w could reverse the thermosensitivity of these bacteria in vivo (17).…”

“…Heterologous complementation studies have revealed that replacing the bacterial J-domain of a thermosensitive E. coli strain (OD259) with the J-domain from PFD0462w could reverse the thermosensitivity of these bacteria in vivo (17). This indicated that these plasmodial J-domains were able to specifically interact with E. coli Hsp70 (DnaK).…”

The Druggable Antimalarial Target PfDXR: Overproduction Strategies and Kinetic Characterization

“…Amino acid codons used most frequently in P. falciparum are different to those used by other organisms [10]. Experimental strategies that have been adopted in order to overcome these obstacles include: rare codon tRNA supplementation [11], codon optimization [10,12] and codon harmonization [13].…”

“…This strategy was used successfully for the production of full-length P. falciparum heat shock protein 70 (PfHsp70) [14]. Codon optimization is an experimental strategy in which the coding region of a target protein is designed to include the most frequently used codons of the host cell [12]. However, the expression of many malaria proteins was not improved through codon optimization [15].…”