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Published on: September 21, 2011
Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein
V Prapapanich1, S Chen, S C Nair
1Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198, USA.
Insights
Researchers identified a novel 48-kDa protein (p48) that associates with the progesterone receptor. This protein is a component of the cytoplasmic molecular chaperone machinery, potentially involved in cellular processes.
Area of Science:
- Molecular Biology
- Cell Biology
- Protein Biochemistry
Background:
- The progesterone receptor is a key regulator of gene expression.
- Understanding the assembly and regulation of the progesterone receptor complex is crucial for comprehending cellular signaling pathways.
Purpose of the Study:
- To isolate and characterize a 48-kDa protein (p48) that transiently associates with the progesterone receptor during cell-free assembly.
- To determine the function and cellular localization of p48.
Main Methods:
- Two-dimensional gel electrophoresis for protein separation.
- Antipeptide antiserum and monoclonal antibodies for p48 detection and purification.
- cDNA cloning and sequencing from a HeLa expression library.
- Northern blot analysis for tissue expression.
- In vitro expression and immunoprecipitation assays.
Main Results:
- A 48-kDa protein (p48) was isolated and its cDNA was cloned and sequenced, revealing a 369-amino acid protein.
- Northern blot analysis indicated ubiquitous expression of p48 in human tissues.
- In vitro studies confirmed p48's association with the progesterone receptor and its interaction with heat shock proteins (hsp70, hsp90) and p60.
Conclusions:
- p48 is a novel component of the cytoplasmic molecular chaperone machinery.
- p48 likely plays a role in the assembly or function of the progesterone receptor complex.
Abstract:
A 48-kDa protein (p48) that transiently associates with progesterone receptor during cell-free assembly in rabbit reticulocyte lysate was isolated by two-dimensional gel separation. Tryptic peptide sequences were generated and used to develop an antipeptide antiserum recognizing p48 by Western immunostaining, and this antiserum was used to monitor purification of native p48 from reticulocyte lysate. Eight mouse monoclonal antibodies capable of immunoprecipitating vertebrate p48 were generated. These monoclonal antibodies served as probes to clone ten p48 cDNAs from a HeLa cDNA expression library. One of the cloned cDNAs was sequenced in its entirety and codes for a 369-amino acid protein (calculated Mr = 41,324). Northern blot analysis of RNA from multiple human tissues suggest that p48 may be ubiquitously expressed. Expression of human p48 cDNA in vitro yielded a product that comigrated with rabbit p48 by SDS-PAGE and associated with progesterone receptor in a similar manner. Immunoprecipitation of p48 complexes revealed a common association of p48 with hsp70 and, to a lesser extent, with hsp90 and p60. Thus, it appears that p48 is a novel component of the cytoplasmic molecular chaperone machinery.
