Marta E. Hallak

RELEASE OF TYROSINE FROM TYROSINATED TUBULIN. SOME COMMON FACTORS THAT AFFECT THIS PROCESS AND THE ASSEMBLY OF TUBULIN

An enzyme system present in the soluble fraction of rat brain homogenate catalyzes the incorporation of tyrosine into the carboxyl end of the a-subunit of tubulin [ 1,2] . The system does not require nucleic acids [3]. Results from studies on the incorporation of [r4C] tyrosine into brain tubulin in animals whose protein synthesis was inhibited by cycloheximide indicated that a similar system operates in vivo [4]. The present work deals with an activity found in the soluble fraction of rat brain that determines the release of the C-terminal tyrosine from tyrosinated tubulin. Every previously known inhibitor or activator of assembly of tubulin we tested also affected the release of tyrosine when assayed in similar conditions. The relationship of the activity reported here with a similar one previously described [S] for which was claimed that ADP and Pi were required is discussed.

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [14C]tyrosine into the C‐terminal position of α‐tubulin of rat brain cytosol was 10‐fold higher for non‐assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non‐assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.

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.

Myelin basic protein functions as a microtubule stabilizing protein in differentiated oligodendrocytes.

Myelin basic protein (MBP) is an oligodendrocyte‐specific protein essential for oligodendrocyte morphogenesis at late stages of cell differentiation. There is evidence that the morphogenetic function of MBP is mediated by MBP interaction with the cytoskeleton. Thus, an MBP/cytoplasmic microtubule association has been reported, and MBP has Ca2+/calmodulin‐regulated microtubule cold‐stabilizing activity in vitro. However, the unambiguous demonstration of a microtubule‐stabilizing activity for MBP in cells has been difficult because oligodendrocytes contain variants of STOP (stable tubule only polypeptide) proteins, which are responsible for microtubule cold stability in different cell types. Herein, we have used genetic mouse models and RNA interference to assay independently the microtubule cold‐stabilizing activities of MBP and of STOP in developing oligodendrocytes. In wild‐type oligodendrocytes, microtubules were cold stable throughout maturation, which is consistent with the presence of STOP proteins from early stages of differentiation. In contrast, in oligodendrocytes from STOP‐deficient mice, microtubules were cold labile in the absence of MBP expression or when MBP expression was restricted to the cell body and became stable in fully differentiated oligodendrocytes, where MBP is expressed in cell extensions. The suppression of MBP by RNA interference in STOP‐deficient oligodendrocytes suppressed microtubule cold stability. Additionally, STOP suppression in oligodendrocytes derived from shiverer mice that lack MBP led to the complete suppression of microtubule cold stability at all stages of cell differentiation. These results demonstrate that both STOP and MBP function as microtubule‐stabilizing proteins in differentiating oligodendrocytes and could be important for the morphogenetic function of MBP.

Astrocytes and oligodendrocytes express different STOP protein isoforms

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N‐STOP and E‐STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F‐STOP (42 kDa) and two minor variants of 48 and 89 kDa. N‐ and E‐STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F‐STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O‐STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A‐STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule‐stabilizing capacities.

The arginylation-dependent association of calreticulin with stress granules is regulated by calcium

Post-translational modifications of proteins are important for the regulation of cell functions; one of these modifications is post-translational arginylation. In the present study, we show that cytoplasmic CRT (calreticulin) is arginylated by ATE1 (arginyl-tRNA protein transferase). We also show that a pool of CRT undergoes retrotranslocation from the ER (endoplasmic reticulum) to the cytosol, because in CRT-knockout cells transfected with full-length CRT (that has the signal peptide), cytoplasmic CRT appears as a consequence of its expression and processing in the ER. After the cleavage of the signal peptide, an N-terminal arginylatable residue is revealed prior to retrotranslocation to the cytoplasm where arginylation takes place. SGs (stress granules) from ATE1-knockout cells do not contain CRT, indicating that CRT arginylation is required for its association to SGs. Furthermore, R-CRT (arginylated CRT) in the cytoplasm associates with SGs in cells treated with several stressors that lead to a reduction of intracellular Ca2+ levels. However, in the presence of stressors that do not affect Ca2+ levels, R-CRT is not recruited to these loci despite the fact that SGs are formed, demonstrating Ca2+-dependent R-CRT association to SGs. We conclude that post-translational arginylation of retrotranslocated CRT, together with the decrease in intracellular Ca2+, promotes the association of CRT to SGs.

Protein Arginylation in Rat Brain Cytosol: A Proteomic Analysis

Arginine can be post-translationally incorporated from arginyl-tRNA into the N-terminus of soluble acceptor proteins in a reaction catalyzed by arginyl-tRNA protein transferase. In the present study, several soluble rat brain proteins that accepted arginine were identified after arginine incorporation by two dimensional electrophoresis and mass spectrometry. They were identified as: contrapsin-like protease inhibitor-3, α-1-antitrypsin, apolipoprotein E, hemopexin, calreticulin and apolipoprotein A-I. All of these proteins shared a signal sequence for the translocation of proteins across endoplasmic reticulum membranes. After losing the signal peptide, these proteins expose amino acids described as compatible for post-translational arginylation. Although the enzymatic system involved in arginylation is confined mainly in cytosol and nucleus, all the substrates described herein enter to the exocytic pathway co-translationally. Therefore, we postulate that the substrates for arginylation could reach the cytosol by retro-translocation and be then arginylated.

Posttranslational arginylation of soluble rat brain proteins after whole body hyperthermia.

We have previously reported the posttranslational addition of [14C]‐arginine in the N‐terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule‐associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]‐arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]‐arginine into proteins was observed in comparison with animals maintained at 28°C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]‐arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]‐methionine‐labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins. J. Neurosci. Res. 56:85–92, 1999. 

Arginylated Calreticulin at Plasma Membrane Increases Susceptibility of Cells to Apoptosis

Background: Calreticulin retrotranslocated from the endoplasmic reticulum to the cytoplasm is post-translationally arginylated associates to stress granules following stress that decrease Ca2+. Results: Arginylated calreticulin reaches the plasma membrane as a response to stress. Conclusion: Arginylation confers to calreticulin a function as one of the preapoptotic signals of the cells. Significance: Arginylated calreticulin is a novel factor involved in stress-induced apoptosis. Post-translational modifications of proteins are important for the regulation of cell fate and functions; one of these post-translational modifications is arginylation. We have previously established that calreticulin (CRT), an endoplasmic reticulum resident, is also one of the arginylated substrates found in the cytoplasm. In the present study, we describe that arginylated CRT (R-CRT) binds to the cell membrane and identified its role as a preapoptotic signal. We also show that cells lacking arginyl-tRNA protein transferase are less susceptible to apoptosis than wild type cells. Under these conditions R-CRT is present on the cell membrane but at early stages is differently localized in stress granules. Moreover, cells induced to undergo apoptosis by arsenite show increased R-CRT on their cell surface. Exogenously applied R-CRT binds to the cell membrane and is able to both increase the number of cells undergoing apoptosis in wild type cells and overcome apoptosis resistance in cells lacking arginyl-tRNA protein transferase that express R-CRT on the cell surface. Thus, these results demonstrate the importance of surface R-CRT in the apoptotic response of cells, implying that post-translational arginylation of CRT can regulate its intracellular localization, cell function, and survival.

Calreticulin-dimerization induced by post-translational arginylation is critical for stress granules scaffolding.

Protein arginylation mediated by arginyl-tRNA protein transferase is a post-translational modification that occurs widely in biology, it has been shown to regulate protein and properties and functions. Post-translational arginylation is critical for embryogenesis, cardiovascular development and angiogenesis but the molecular effects of proteins arginylated in vivo are largely unknown. In the present study, we demonstrate that arginylation reduces CRT (calreticulin) thermostability and induces a greater degree of dimerization and oligomerization. R-CRT (arginylated calreticulin) forms disulfide-bridged dimers that are increased in low Ca(2+) conditions at physiological temperatures, a similar condition to the cellular environment that it required for arginylation of CRT. Moreover, R-CRT self-oligomerizes through non-covalent interactions that are enhanced at temperatures above 40 °C, condition that mimics the heat shock treatment where R-CRT is the only isoespecies of CRT that associates in cells to SGs (stress granules). We show that in cells lacking CRT the scaffolding of larger SGs is impaired; the transfection with CRT (hence R-CRT expression) restores SGs assembly whereas the transfection with CRT mutated in Cys146 does not. Thus, R-CRT disulfide-bridged dimers (through Cys146) are essential for the scaffolding of larger SGs under heat shock, although these dimers are not required for R-CRT association to SGs. The alteration in SGs assembly is critical for the normal cellular recover of cells after heat induced stress. We conclude that R-CRT is emerging as a novel protein that has an impact on the regulation of SGs scaffolding and cell survival.