Carlo Turano

Proteins of the PDI family: Unpredicted non-ER locations and functions

Protein disulfide isomerases (PDIs) constitute a family of structurally related enzymes which catalyze disulfide bonds formation, reduction, or isomerization of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER). They act also as chaperones, and are, therefore, part of a quality‐control system for the correct folding of the proteins in the same subcellular compartment. While their functions in the ER have been thoroughly studied, much less is known about their roles in non‐ER locations, where, however, they have been shown to be involved in important biological processes. At least three proteins of this family from higher vertebrates have been found in unusual locations (i.e., the cell surface, the extracellular space, the cytosol, and the nucleus), reached through an export mechanism which has not yet been understood. In some cases their function in the non‐ER location is clearly related to their redox properties, but in most cases their mechanism of action has still to be disclosed, although their propensity to associate with other proteins or even with DNA might be the main factor responsible for their activities.

Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA–protein cross‐linking experiments performed with two different cross‐linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR‐like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus. J. Cell. Biochem. 85: 325–333, 2002.

ERp57/GRP58: A protein with multiple functions

The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.

Multiple forms of supernatant glutamate-aspartate transaminase from pig heart.

Abstract Holo glutamate-aspartate transaminase from pig heart has been purified by Lis (1958) and by Jenkins et al . (1959) . These preparations contain essentially the supernatant enzyme ( Wada and Morino, 1964 ). Preparations homogeneous in the ultracentrifuge and in free boundary electrophoresis had an O.D. 280mμ O.D. 362mμ ratio of 10 at pH 9; this property was often used as a routine criterium for purity. Hetereogeneity of preparations which met these specifications has recently been reported ( Jenkins,1965 ). It is the purpose of the present communication to show that such preparations contain multiple forms of the supernatant enzyme differring in specific activity, spectrophotometric properties and motility on starch gel electrophoresis.

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose‐regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress‐response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non‐coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non‐coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress‐response genes. J. Cell. Physiol. 210: 343–351, 2007.

Binding of the protein disulfide isomerase isoform ERp60 to the nuclear matrix-associated regions of DNA.

Protein ERp60, previously found in the internal nuclear matrix in chicken liver nuclei, is a member of the protein disulfide isomerase family. It binds DNA and double helical polynucleotides in vitro with a preferential recognition toward the matrix‐associated regions of DNA and poly(dA)·poly(dT), and its binding is inhibited by distamycin. ERp60 can be cross‐linked chemically to DNA in the intact nuclei, suggesting that its association with DNA is present in vivo. As a whole, these results indicate that ERp60 is a component of the subset of nuclear matrix proteins that are responsible for the attachment of DNA to the nuclear matrix and for the formation of DNA loops. A distinctive feature of this protein, which has two thioredoxin‐like sites, is that its affinity to poly(dA)·poly(dT) is strongly dependent on its redox state. Only its oxidized form, in fact, does it bind poly(dA)·poly(dT). The hypothesis can be made that through the intervention of ERp60, the redox state of the nucleus influences the formation or the stability of some selected nuclear matrix–DNA interactions. J. Cell. Biochem. 72:528–539, 1999.

On the mechanism of action of glutamic-aspartic transaminase: intermediate steps in the reaction.

Abstract Substrate amounts of highly purified GAT react with glutamate, aspartate and, at a much slower rate, with alanine, but not with glycine, arginine, serine and isoleucine, to give the corresponding ketoacids. The absorption spectrum of the enzyme changes during the reaction and the coenzyme passes from the aldehydic (enzyme-PLP) to the aminic (enzymic-PMP) form. Enzyme-PMP was isolated from the reaction mixture and its spectrum was studied at different pH values. The coenzyme/protein ratio and the specific activity of enzyme-PMP were the same as those of enzyme-PLP. The aminic form of the enzyme reacts with ketoglutarate, oxalacetate and, at a slower rate, with pyruvate, to give the corresponding amino acids and the aldehydic form of the enzyme.

A reverse-phase HPLC method for cAMP phosphodiesterase activity

A simple and fast method based on reverse-phase HPLC has been developed for measuring the activity of cAMP phosphodiesterase. It allows quantitation of product and substrate in less than 10 min. The sensitivity (1*10(-11) mol AMP), the accurate evaluation of nucleotides, the unequivocal analysis of product, and the reproducibility of the system, make this method suitable for the evaluation of cAMP phosphodiesterase in biological material, at different levels of purification, and also in kinetic studies.

Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line

Chromatin immunoprecipitation in M14 melanoma cells showed that the protein ERp57 (endoplasmic reticulum protein 57) binds to DNA in the proximity of STAT3 in a subset of STAT3-regulated genes. In the same cells, IL-6 induced a significant increase of the expression of one of these genes, i.e. CRP. Upon depletion of ERp57 by RNA interference, the phosphorylation of STAT3 on tyrosine 705 was decreased, and the IL-6-induced activation of CRP expression was completely suppressed. In vitro experiments showed that ERp57 is also required for the binding of STAT3 to its consensus sequence on DNA. Thus ERp57, previously shown to associate with STAT3 in the cytosol and in the nuclear STAT3-containing enhanceosome, is a necessary cofactor for the regulation of at least a subset of STAT3-dependent genes, probably intervening both at the site of STAT3 phosphorylation and at the nuclear level.

Crosslinking of nuclear proteins to DNA by cis-diammminedichloroplatinum in intact cells Involvement of nuclear matrix proteins

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis‐diamminedichloroplatinum on intact chicken liver cells and analyzed by two‐dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.