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Alternative expression of ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, is a neuron survival factor, was studied in the current work. Computer analysis showed that Cp-mRNA isoform, coding for 109 kDa polypeptide, can be formed as a result of the transcription from the alternative promoter in 3'-region of intron 2 of rat Cp gene. Alternative Cp form starts with 25 amino acid residues sequence, coded with intron 2 region. It is followed by amino acid sequence of the main Cp isoform starting from Gly 113. In silico data were experimentally confirmed using RT-PCR. It was demonstrated that the predicted mRNA was generally localized in liver and brain cells of adult rats. Direct sequencing of the obtained PCR-product showed the entire coincidence of the real and predicted mRNAs. It was in vitro showed that approximately 110 kDa Cp-like protein was completed and accumulated in the absence of mitochondria. This protein is transferred into the isolated mitochondria in the reconstructed system. Transport is energy-dependent, it is not accompanied with the shortening of Cp polypeptide length and needs the presence of cytosolic factors. Probably import is determined by the inner protein mitochondria import signal with amino acid sequence KVVYREFTDSTFRE, located in 756-769 region of mature Cp. Possible role of Cp in iron metabolism in mitochondria is under discussion.

Using immunoblotting method it was found out that ceruloplasmin (Cp) polypeptides are revealed in mitochondria of rats, isolated from brain, liver, testicles and mammary gland. Cp is localized in matrix and inner membranes of mitochondria. Its molecular weight corresponds to the non-glycosilated form of the protein. Computer analysis did not reveal any sequences homologous to the signal peptide for mitochondria protein import (SPMPI) in the rat chromosomal Cp gene. However the analysis of homologous to Cp sequences, presented in databases, detected SPMPI in the human processed Cp pseudogene. Cp pseudogene region of 984 nucleotides long is translated in the only reading frame to the polypeptide of 328 a.a. long including 66 a.a of SPMPI at N-terminus. The predicted protein with the exception of SPMPI is identical to the corresponding Cp fragment at 92%, it has 20 amino acid substitutions, 8 of which are significant. His-X-His motif, typical for copper containing ferroxidases, is located in the centre of a molecule. Potential copper-binding site appears as a result of proline to histidine substitution at 923 position along Cp sequence. The product of Cp pseudogene transcription was detected in the human cultured cells of HepG2 and HuTu 80 cell lines using RT-PCR strategy. 30 kDa polypeptide that interacts with human Cp antibodies was found in mitochondria of HuTu 80 cells. The possible biological role of mitochondrial Cp is under discussion.

A study was made of the expression of the ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, acts as a neuron survival factor. Computer analysis predicted an alternative promoter in the 3′-terminal region of intron 2 of the rat Cp gene and showed that transcription from this promoter potentially yields an mRNA coding for a 109-kDa polypeptide. The alternative Cp form starts with a region of 25 amino acid residues encoded by a part of intron 2. From Gly113, its sequence is identical to that of the major Cp form. The in silico data were confirmed experimentally by RT-PCR. The predicted mRNA was found predominantly in the liver and brain of adult rats. Direct sequencing of the PCR product demonstrated the complete identity of the predicted and real mRNA sequences. It was shown in vitro that a Cp-like protein of about 110 kDa is synthesized in the cytosol and accumulates in the absence of mitochondria. The protein was transferred into isolated mitochondria in a reconstructed system. Transport was energy-dependent, was associated with no change in Cp polypeptide length, and required cytosolic factors. Mitochondrial import of the Cp form was probably determined by an internal mitochondrial localization signal, KVVYREFTDSTFRE, corresponding to region 756–769 of mature Cp. The possible role of Cp in mitochondrial iron metabolism is discussed. [ABSTRACT FROM AUTHOR]

Presents a study which examined the expression of ceruloplasmin (Cp) pseudogene in cultured human cells. Purpose and function of Cp in the body; Information on the process of transporting iron in cell compartments; Analysis of Cp-like protein in mammalian mitochondria.