Irene Graziani

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.

The intracellular domain of Notch ligand Delta1 induces cell growth arrest

Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non‐proliferating senescent‐like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by co‐expression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

Novel Cross-Talk between Three Cardiovascular Regulators: Thrombin Cleavage Fragment of Jagged1 Induces Fibroblast Growth Factor 1 Expression and Release

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular domain. The sJ1 form produced as a result of thrombin cleavage inhibits Notch-mediated CBF1/Suppressor of Hairless [(Su(H)]/Lag-1-dependent transcription and induces FGF1 expression and release. The overexpression of Jagged1 in PAR1 null cells results in a rapid thrombin-induced export of FGF1. These data demonstrate the existence of novel cross-talk between thrombin, FGF, and Notch signaling pathways, which play important roles in vascular formation and remodeling.

Protein folding does not prevent the nonclassical export of FGF1 and S100A13.

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.

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.