Characterization Of HDJC9,a Novel Nucleus-localized Secretory Human Type C DnaJ/Hsp40 Member Interacting With And Cochaperoning Hsp70

Molecular chaperones or heat shock proteins (Hsp) are a set of conserved protein families occurring in all organisms and are indispensable for cell survival under heat stress or other stress conditions with diverse functions, including mediating protein folding and degradation, transport across cellular membranes, and assembly into macromolecular structures. In general, five classes of chaperone proteins have been distinguished: Hsp70, Hsp60, Hsp90, HsplOO and small Hsps according to their molecular weight. Hsp70 are monomeric proteins that bind to unfolded, mainly hydrophobic segments of substrate proteins, in an ATP-regulated manner. Escherichia coli DnaJ, the progenitor of the eukaryotic Hsp40 family, is a cofactor of DnaK, the bacterial hsp70 homologue, and can stimulate the ATPase activity of DnaK and thus regulate the binding capacity of DnaK to substrate proteins. Here we report the cloning and characterization of a novel human type C DnaJ homologue HDJC9 containing a typical N-terminal J domain. Part I Cloning, sequence analysis of HDJC9The cDNA sequence encoding HDJC9 was directly isolated from dendritic cell cDNA library by large-scale sequencing. The cDNA is of 1431bp, contains a complete open reading fame (ORF) of 783bp, which potentially encodes a 260-residue protein and is deposited in GenBank under accession number AF327347. Downstream the stop codon (TAA), we found a Cozak signature and a poly A tail, indicating that the cDNA encoding HDJC9 was of full-length. Homology analysis was performed using BLAST program. At protein level, it shares highest homology with BAB85076.1/NP₀56005.1/Q8WXX5 (100%) from Homo sapiens and was similar to XP₅64165.1 from Canus familiaris (88% identity), JC7707 from Rattus norvegicus (84% identity) and NP₅98842.1/AAH23787.1 from Mus Musculus (82% identity). HDJC9 shows more than 84% identity and 90% similarity with a rat clone(XM₃44286) also encoding a 260-residue protein designated as JDD1 (J domain of DnaJ-like protein), which may be the rat homologue of human HDJC9. By alignment of HDJC9 protein with homologues, we found that HDJC9 contained an N-terminal J domain (12-98 aa) about 87 residues and a 162-residue C-terminal domain with unknown functions, but not the G/F-rich domain and zinc finger-like domain. Therefore, we designated this novel DnaJ protein as HDJC9, human DnaJ/subfamily C/member 9 protein, using nomenclature principle suggested by HUGO. HDJC9-like proteins can be found in various species ranging from Caenorhabditis elegans (BC254216) to Drosophila melanogaster (XM₁41795) to Xenopus laevis (BC090203) to Danio rerio (NM₀01002433) to Homo sapiens. Evolutionally, HDJC9 was close to HDJC11/FLJ10737, RDJC5, MDJC1, MDJC6, HDJC1O/Erdj5/JPD1, HDJC7/Tpr2, HDJB2/HSJ1, HDJB7 andHDJB6/Hdj-l/MRJ/HSJ-2. By Blasting genome database, we found that HDJC9 was overlapping with UniGene Cluster Hs. 523037 (10q22.2) and that HDJC9 gene overlapped 4.6 kb in the human genome containing 5 exons. Part II Expression pattern, nuclear localization and heat shock-induced secretion of HDJC9By RT-PCR, we have demonstrated the mRNA expression of HDJC9 in some solid tumor cells, including breast adenocarcinoma MCF-7, glioblasoma A172, cerix epitheloid carcinoma HeLa, hepatoma SMMC7721, prostate adenocarcinoma PC-3, HT29 and ovary adenocarcinoma CaoV-3 cells. However, the Colon adenocarcinoma cell lines Lo Vo and lung carcinoma A549 do not express HDJC9. HDJC9 mRNA was also detected in hematopoietic tumor cells, including histiocytic lymphoma U937, chronic myelogenous leukemia K562, Burkitt's lymphoma Daudi cells. The mRNA expression pattern of HDJC9 in normal human adult tissues was examined by Northern. The result showed that HDJC9 had a wide distribution in normal human tissues, including heart, spleen, kidney, placenta, lung, liver, ovary, testis, skeletal muscle, colon, prostate, thymus, pancreas, but no expression was detected in brainand small intestine.Sequence and homology analysis has suggested that HDJC9 be a member of DnaJ family. To investigate the subcellular localiza...