The GTPase Rab21 is required for neuronal development and migration in the cerebral cortex.

Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.

Assaying the Posttranslational Arginylation of Proteins in Cultured Cells.

To evaluate the posttranslational arginylation of proteins in vivo, we describe a protocol for studying the 14C-Arg incorporation into proteins of cells in culture. The conditions determined for this particular modification contemplate both the biochemical requirements of the enzyme ATE1 and the adjustments that allowed the discrimination between posttranslational arginylation of proteins and de novo synthesis. These conditions are applicable for different cell lines or primary cultures, representing an optimal procedure for the identification and the validation of putative ATE1 substrates.

Arginylated Calreticulin Increases Apoptotic Response Induced by Bortezomib in Glioma Cells.

After retrotranslocation from the endoplasmic reticulum to the cytoplasm, calreticulin is modified by the enzyme arginyltransferase-1 (ATE1). Cellular levels of arginylated calreticulin (R-CRT) are regulated in part by the proteasomal system. Under various stress conditions, R-CRT becomes associated with stress granules (SGs) or reaches the plasma membrane (PM), where it participates in pro-apoptotic signaling. The mechanisms underlying the resistance of tumor cells to apoptosis induced by specific drugs remain unclear. We evaluated the regulatory role of R-CRT in apoptosis of human glioma cell lines treated with the proteasome inhibitor bortezomib (BT). Two cell lines (HOG, MO59K) displaying distinctive susceptibility to apoptosis induction were studied further. BT efficiency was found to be correlated with a subcellular distribution of R-CRT. In MO59K (apoptosis-resistant), R-CRT was confined to SGs formed following BT treatment. In contrast, HOG (apoptosis-susceptible) treated with BT showed lower SG formation and higher levels of cytosolic and PM R-CRT. Increased R-CRT level was associated with enhanced mobilization of intracellular Ca2+ and with sustained apoptosis activation via upregulation of cell death receptor DR5. R-CRT overexpression in the cytoplasm of MO59K rendered the cells susceptible to BT-induced, DR5-mediated cell death. Our findings suggest that R-CRT plays an essential role in the effect of BT treatment on tumor cells and that ATE1 is a strong candidate target for future studies of cancer diagnosis and therapy.

Relevance of protein-protein interactions on the biological identity of nanoparticles.

Considering that the use of nanoparticles (NPs) as carriers of therapeutic or theranostic agents has increased in the last years, it is mandatory to understand the interaction between NPs and living systems. In contact with biological fluids, the NPs (synthetic identity) are covered with biomolecules that form a protein corona, which defines the biological identity. It is well known that the protein corona formation is mediated by non-specific physical interactions, but protein-protein interactions (PPI), involving specific recognition sites of the polypeptides, are also involved. This work explores the relationship between the synthetic and biological identities of layered double hydroxides nanoparticles (LDH-NPs) and the effect of the protein corona on the cellular response. With such a purpose, the synthetic identity was modified by coating LDH-NPs with either a single protein or a complex mixture of them, followed by the characterization of the protein corona formed in a commonly used cell culture medium. A proteomic approach was used to identify the protein corona molecules and the PPI network was constructed with a novel bioinformatic tool. The coating on LDH-NPs defines the biological identity in such a way that the composition of the protein corona as well as PPI are changed. Electrostatic interactions appear not to be the only driving force regulating the interactions between NPs, proteins and cells since the specific recognition also play a fundamental role. However, the biological identity of LDH-NPs does not affect the interactions with cells that shows negligible cytotoxicity and high internalization levels.

Post-translational protein arginylation in the normal nervous system and in neurodegeneration.

Post-translational arginylation of proteins is an important regulator of many physiological pathways in cells. This modification was originally noted in protein degradation during neurodegenerative processes, with an apparently different physiological relevance between central and peripheral nervous system. Subsequent studies have identified a steadily increasing number of proteins and proteolysis-derived polypeptides as arginyltransferase (ATE1) substrates, including ß-amyloid, α-synuclein, and TDP43 proteolytic fragments. Arginylation is involved in signaling processes of proteins and polypeptides that are further ubiquitinated and degraded by the proteasome. In addition, it is also implicated in autophagy/lysosomal degradation pathway. Recent studies using mutant mouse strains deficient in ATE1 indicate additional roles of this modification in neuronal physiology. As ATE1 is capable of modifying proteins either at the N-terminus or middle-chain acidic residues, determining which proteins function are modulated by arginylation represents a big challenge. Here, we review studies addressing various roles of ATE1 activity in nervous system function, and suggest future research directions that will clarify the role of post-translational protein arginylation in brain development and various neurological disorders. Arginyltransferase (ATE1), the enzyme responsible for post-translational arginylation, modulates the functions of a wide variety of proteins and polypeptides, and is also involved in the main degradation pathways of intracellular proteins. Regulatory roles of ATE1 have been well defined for certain organs. However, its roles in nervous system development and neurodegenerative processes remain largely unknown, and present exciting opportunities for future research, as discussed in this review.

Assaying the Posttranslational Arginylation of Proteins in Cultured Cells.

To evaluate the posttranslational arginylation of proteins in vivo, we describe a protocol for studying the (14)C-Arg incorporation into proteins of cells in culture. The conditions determined for this particular modification contemplate both the biochemical requirements of the enzyme ATE1 and the adjustments that allowed the discrimination between posttranslational arginylation of proteins and de novo synthesis. These conditions are applicable for different cell lines or primary cultures, representing an optimal procedure for the identification and the validation of putative ATE1 substrates.

Post-translational arginylation of calreticulin: a new isospecies of calreticulin component of stress granules.

Post-translational arginylation consists of the covalent union of an arginine residue to a Glu, Asp, or Cys amino acid at the N-terminal position of proteins. This reaction is catalyzed by the enzyme arginyl-tRNA protein transferase. Using mass spectrometry, we have recently demonstrated in vitro the post-translational incorporation of arginine into the calcium-binding protein calreticulin (CRT). To further study arginylated CRT we raised an antibody against the peptide (RDPAIYFK) that contains an arginine followed by the first 7 N-terminal amino acids of mature rat CRT. This antibody specifically recognizes CRT obtained from rat soluble fraction that was arginylated in vitro and also recognizes endogenous arginylated CRT from NIH 3T3 cells in culture, indicating that CRT arginylation takes place in living cells. Using this antibody we found that arginylation of CRT is Ca2+-regulated. In vitro and in NIH 3T3 cells in culture, the level of arginylated CRT increased with the addition of a Ca2+ chelator to the medium, whereas a decreased arginine incorporation into CRT was found in the presence of Ca2+. The arginylated CRT was observed in the cytosol, in contrast to the non-arginylated CRT that is in the endoplasmic reticulum. Under stress conditions, arginylated CRT was found associated to stress granules. These results suggest that CRT arginylation occurs in the cytosolic pool of mature CRT (defined by an Asp acid N-terminal) that is probably retrotranslocated from the endoplasmic reticulum.

Re-examination of the post-translational arginylated protein of 125-kD initially identified as N-STOP.

Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP). This was inferred from its electrophoretic mobility (125 kD) and because it was immunoprecipitated with a monoclonal antibody against the N-STOP. However, this hypothesis is not supported by our recent results. Herein, we found that rat N-STOP interacts with Ca(2+)-calmodulin, whereas the 125-kD [14C]-arginylated protein does not. The 125-kD [14C]-arginylated protein from rat brain is separated from the N-STOP by two-dimensional electrophoresis, and it is not recognized by a STOP monoclonal antibody. Mouse brain contains N-STOP, which migrates as a protein of 116 kD and could not be labeled by the post-translational incorporation of [14C]-arginine. The 125-kD [14C]-arginylated protein appears in wild-type as well as in STOP knock out mice. Based on these results, we conclude that the 125-kD arginylated protein is different from N-STOP.