Cinzia Bagala

Copper Induces the Assembly of a Multiprotein Aggregate Implicated in the Release of Fibroblast Growth Factor 1 in Response to Stress

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.

Copper chelation represses the vascular response to injury

The induction of an acute inflammatory response followed by the release of polypeptide cytokines and growth factors from peripheral blood monocytes has been implicated in mediating the response to vascular injury. Because the Cu2+-binding proteins IL-1α and fibroblast growth factor 1 are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu2+ to facilitate the formation of S100A13 heterotetrameric complexes and these signal peptideless polypeptides have been implicated as regulators of vascular injury in vivo, we examined the ability of Cu2+ chelation to repress neointimal thickening in response to injury. We observed that the oral administration of the Cu2+ chelator tetrathiomolybdate was able to reduce neointimal thickening after balloon injury in the rat. Interestingly, although immunohistochemical analysis of control neointimal sections exhibited prominent staining for MAC1, IL-1α, S100A13, and the acidic phospholipid phosphatidylserine, similar sections obtained from tetrathiomolybdate-treated animals did not. Further, adenoviral gene transfer of the IL-1 receptor antagonist during vascular injury also significantly reduced the area of neointimal thickening. Our data suggest that intracellular copper may be involved in mediating the response to injury in vivo by its ability to regulate the stress-induced release of IL-1α by using the nonclassical export mechanism employed by human peripheral blood mononuclear cells in vitro.

The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export.

The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.

S100A13 mediates the copper-dependent stress- induced release of IL-1α from both human U937 and murine NIH 3T3 cells

Copper is involved in the promotion of angiogenic and inflammatory events in vivo and, although recent clinical data has demonstrated the potential of Cu2+ chelators for the treatment of cancer in man, the mechanism for this activity remains unknown. We have previously demonstrated that the signal peptide-less angiogenic polypeptide, FGF1, uses intracellular Cu2+ to facilitate the formation of a multiprotein aggregate that enables the release of FGF1 in response to stress and that the expression of the precursor form but not the mature form of IL-1α represses the stress-induced export of FGF1 from NIH 3T3 cells. We report here that IL-1α is a Cu2+-binding protein and human U937 cells, like NIH 3T3 cells, release IL-1α in response to temperature stress in a Cu2+-dependent manner. We also report that the stress-induced export of IL-1α involves the intracellular association with the Cu2+-binding protein, S100A13. In addition, the expression of a S100A13 mutant lacking a sequence novel to this gene product functions as a dominant-negative repressor of IL-1α release, whereas the expression of wild-type S100A13 functions to eliminate the requirement for stress-induced transcription. Lastly, we present biophysical evidence that IL-1α may be endowed with molten globule character, which may facilitate its release through the plasma membrane. Because Cu2+ chelation also represses the release of FGF1, the ability of Cu2+ chelators to potentially serve as effective clinical anti-cancer agents may be related to their ability to limit the export of these proinflammatory and angiogenic signal peptide-less polypeptides into the extracellular compartment.

S100A13 (S100 calcium binding protein A13)

Review on S100A13 (S100 calcium binding protein A13), with data on DNA, on the protein encoded, and where the gene is implicated.

CHAPTER 327 – Nonclassical Pathways of Protein Export

One of the earliest studies of nonclassical protein release was devoted to secretory transglutaminase (TO), an enzyme that cross-links extracellular polypeptides and is released into the extracellular space by cells of the prostate. The majority of proteins that are destined to be secreted are usually translated with an NH2-terminal cleavable signal peptide sequence. This structure is rich in hydrophobic amino acid residues that enable the polypeptide to dock and insert into the lumen of the endoplasmic reticulum for its further transport to the Golgi apparatus and eventual secretion as an intravesicular polypeptide. However, there are groups of extracellular proteins that neither display a signal peptide sequence in their primary structure nor utilize the classical endoplasmic reticulum (ER)-Golgi pathway for their release. This chapter focuses on the mechanisms of stress-induced release of two signaling proteins belonging to this group, fibroblast growth factor 1 (FGF-1) and interleukin 1ɑ (IL-lɑ). However, before discussing the export of these polypeptides, the literature concerning the nonclassical release of other signal peptide-less proteins is briefly reviewed.