Purification and Characterization of a Novel Stress Protein, the 150-kDa Oxygen-regulated Protein (ORP150), from Cultured Rat Astrocytes and Its Expression in Ischemic Mouse Brain

Abstract

As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons.

Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons

“…We observed detectable levels of this protein only in the presence of L-DOPA regardless of the oxidative stress conditions (Table 2) suggesting that L-DOPA may aid hypoxia condition in cells. It has been shown in rat astrocytes that ORP150 is induced by hypoxia within 24 hours, augmented further during early re-oxygenation, and thereafter decreasing to baseline levels by 24 hours in normoxia [49]. Furthermore, the hypoxia-mediated induction of ORP150 appears specific as stress conditions such as heat shock, H 2 O 2 , cobalt chloride, 2-deoxyglucose, or tunicamycin does not affect ORP150 levels [49].…”

“…It has been shown in rat astrocytes that ORP150 is induced by hypoxia within 24 hours, augmented further during early re-oxygenation, and thereafter decreasing to baseline levels by 24 hours in normoxia [49]. Furthermore, the hypoxia-mediated induction of ORP150 appears specific as stress conditions such as heat shock, H 2 O 2 , cobalt chloride, 2-deoxyglucose, or tunicamycin does not affect ORP150 levels [49]. Interestingly, exposure to dopamine has similar ORP induction effects as hypoxia in PC12 cells [50].…”

Modulation of Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Function by Hypoxia-Upregulated Protein 1

“…Furthermore, HYOU1 is upregulated in some human cancers, including head and neck and breast cancers (34,35). The HYOU1 transcript was originally cloned from astrocytes under hypoxic conditions (36), which makes it a protein relevant to KSHV biology, since hypoxia plays a role in the KSHV life cycle (37). Previous work has indicated that HYOU1 can suppress hypoxia-induced cell death (38) and induce angiogenesis by facilitating VEGF processing (39).…”

Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential

“…GRP170, encoded in humans by HYOU1, is a large HSP70/HSP110-like protein in the ER 184 that is induced by hypoxia 185,186 (see figure).…”