Research reportPresence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain
Introduction
Several polypeptides known as heat- or stress-inducible proteins are expressed in prokaryotes and eukaryotes. They are commonly referred to as heat-shock proteins (Hsps). The Hsps are classified into several groups according to their molecular sizes. The expressions of these proteins are induced by the activation of heat shock elements (HSE) in the promoter region of their genes. HSP70, a homologue of E. coli DnaK protein, has two major members, Hsp70 (p72), an inducible form, and the heat shock cognate protein (Hsc 70, p73), a constitutively expressed form. We refer to the constitutive form of HSP70 as Hsc70 or p73, and the inducible form as p72. We use fully capitalized names to denote the protein family (ex. HSP70) and an initial capital letter for specific family members (ex. Hsc70, Hsp40) according to the recent instruction [14]. The general term HSP70 implies both Hsc70 (p73) and p72. The two proteins are similar with approximately 90% identity in amino acid sequences and mostly indistinguishable biochemical properties except for a slight difference in their electrophoretic mobility [37]. HSP70 is expressed ubiquitously in various tissues, but among adult tissues, only the brain retains high levels of Hsc70 gene expression [13]. The relative levels of Hsp70 mRNA and Hsc70 mRNA are higher in cerebellum than in cerebral cortex and both the transcription and turnover of Hsp70 mRNAs differs between cerebellum and cerebral cortex [30]. The expression of brain HSP70 can be induced by seizure [20]and ischemia 2, 41. Thus, Hsp70 is one of the candidate plasticity-related genes (CPGs) [33].
HSP70 has chaperone activity, which facilitate the repair partially denatured proteins after various stress or assists correct folding of nascent polypeptides [4]. HSP70s in the yeast endoplasmic reticulum and mitochondria are involved in the translocation and targeting of proteins [6]. Furthermore, HSP70 appears to be involved in the transport of cytoplasmic proteins into nucleus, and shuttles between these two compartments 19, 27, 28, 43, 45. HSP70 does not function by itself in vivo and requires assistance by the DnaJ family proteins. HSP70, with the aid of various kinds of DnaJ-like proteins, fulfills various cellular functions 5, 10. All DnaJ-like proteins contain J-domains, by which the proteins interact with HSP70 and stimulate its ATPase activity. Different DnaJ-like proteins have specialized functions, being coupled to HSP70. Thus, the roles of HSP70 and its target molecules are likely to be determined by the types of partner DnaJ homologs [6]or co-compartmentalization of certain types of DnaJ homologs and HSP70s [44]. Ohtsuka et al. [35]have recently identified a novel DnaJ-like protein, Hsp40, in mammalian and avian cells and showed the co-localization of Hsp40 and HSP70 in HeLa cells and various mammalian cells 17, 56.
A detailed localization of HSP70 and its partner chaperones in the brain have not yet been reported. We report here the distribution of Hsc70 and Hsp40 at synaptic sites, and discuss their potential roles in neuronal and synaptic functions.
Section snippets
Materials
Mouse monoclonal anti-p72 antibody (RPN1197) was purchased from Amersham; mouse monoclonal anti-synaptophysin antibody (MAB368) was from Chemicon; anti-PSD-95 antibody was from Transduction Laboratories; anti-rabbit IgG was from Vector Laboratories; Vectastain ABC kit containing anti-rabbit IgG and avidin-biotinylated horseradish peroxidase (HRPO) complex was from Vector Laboratories; HRPO-coupled goat anti-rabbit IgG (H and L) from Calbiochem.; HRPO-coupled goat anti-mouse Ig(G+A+M) from
Identification of HSP70 and Hsp40 in the PSD fraction
We used three different antibodies to HSP70 and an antibody to Hsp40. The validity of these antibodies has been previously reported 17, 56, and reproduced in Western blots using HeLa cells either nontreated or treated at 45°C (Fig. 1). All antibodies showed specific bands with proper molecular sizes, and detected increased expression of HSP70 and Hsp40 after heat-treatment of cells except for the 1B5 antibody which recognizes Hsc70, the constitutive form of HSP70. Thus, we judged that the
Presence of Hsc70 and Hsp40 in the postsynaptic structures
The presence of two kinds of chaperone proteins, Hsc70 and Hsp40, in postsynaptic structures was shown in this paper. Hsc70, a constitutive form of HSP70, was localized at synapses, being present both in the pre- and post-synaptic components but more frequently in postsynaptic. The presence of Hsc70 in postsynaptic sites has been suggested previously, however, its presence in vivo was not addressed. Only the presence of an Hsc70 epitope in the PSD fraction [24]and a short peptide homologous to
Acknowledgements
We thank Dr. P.T. Kelly, University of Texas Medical School, Houston, TX, for his critical reading of the manuscript. This research was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, The Ichiro Kanehara Foundation, and Toyota Physical and Chemical Research Institute.
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