Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library

Abstract

In this manuscript, we describe the generation of a gene library for the expression of HSP110/HSPH, HSP70/HSPA and HSP40/DNAJ members. First, the heat shock protein (HSP) genes were collected from the gene databases and the gene families were analyzed for expression patterns, heat inducibility, subcellular localization, and protein homology using several bioinformatics approaches. These results can be used as a working draft model until data are confirmed by experimental approaches. In addition, we describe the generation of a HSPA/DNAJ overexpression library and tested the effect of different fusion tags on HSPA and DNAJ members using different techniques for measuring chaperone activity. These results show that we have cloned a high-quality heat shock protein expression library containing most members from the HSPH, HSPA, DNAJA and DNAJB families which will be useful for the chaperone community to unravel the function of the highly diverse family of human molecular chaperones.

Fig. 1

Phylograms for the HSPH/A (A), DNAJA (B), DNAJB (C), and DNAJC (D) families. Primary amino acid alignments were performed using the Neighbor-joining algorithm using a Blosum scoring matrix in ClustalX (see “Methods” for details). Bootstrap values are indicated on the branch-points

Fig. 2

Schematic overview of the library construction. (A) The pCDNA5/FRT/TO vector system together with the cloned fusion tags. (B) List of the cloned molecular chaperones. (C) Schematic representation of the FRT locus within the Flp-In T-REx HEK-293 cell line. (D) Primer sequences for the construction of the indicated fusion tags

Fig. 3

The effect of different fusion tags on HSPA1A (A) and (B) or DNAJB1 (C). (A) Luciferase refolding assay using GFP, V5, and untagged HSPA1A versions. Cells were transfected with different tagged versions of HSPA1A together with a plasmid encoding firefly luciferase. HSPA1A expression was induced using tetracycline. The day after transfection, the cells were heated at 37°C or 45°C for 30 min and reincubated for 1 h at 37°C to allow luciferase refolding. Thereafter, cells were lysed and measured for luciferase activity. The percentage of luciferase activity is plotted relative to the activity in unheated control cells (100%). (B) Modulation of tagged HSPA1A versions by BAG-1. Cells were treated as in (A) but also co-transfected with a BAG-1 encoding plasmid as indicated. (C) Filter trap assay showing aggregation of expanded Huntingtin. GFP, V5 and untagged versions of DNAJB1 were used as indicated. Cells were transfected with different tagged versions of DNAJB1 together with a plasmid encoding GFP-tagged Huntingtin containing either 23Q or 74Q. DNAJB1 was induced by tetracycline. Two days after transfection, cells were lysed and the lysates were loaded on to a cellulose acetate membrane. After transblotting, blots were immunostained for GFP to detect aggregated Huntingtin. GFP-tagged DNAJB1 alone did not show any signal on the membrane (not shown)