Metallothionein-3 Is a Component of a Multiprotein Complex in the Mouse Brain

Abstract

Metallothionein (MT)-3, originally called growth inhibitory factor (GIF), was initially identified through its ability to inhibit the growth of neuronal cells in the presence of brain extract. MT-3 is the brain specific isoform of the MT family whose specific biological activity associates it with neurological disorders. Indeed, studies report that MT-3 is decreased by ~30% in brains of patients with Alzheimer disease (AD). Furthermore, many lines of evidence suggest that MT-3 engages in specific protein interactions. To address this, we conducted immunoaffinity chromatography experiments using an immobilized anti-mouse MT-3 antibody. We identified five associated proteins from the pool of sixteen recovered using mass spectrometry and tandem mass spectrometry after in-gel trypsin digestion of bands from the affinity chromatography. The proteins identified were: heat shock protein 84 (HSP84), heat shock protein 70 (HSP70), dihydropyrimidinase-like protein-2 (DRP-2), creatine kinase (CK) and beta-actin. Coimmunoprecipitation experiments, also conducted on whole mouse brain extract using the anti-mouse MT-3 antibody along with commercially available antibodies against HSP84 and CK, confirmed that these three proteins were in a single protein complex. Immunohistochemical experiments were then conducted on the perfused mouse brain that confirmed the in situ colocalization of CK and MT-3 in the hippocampus region. These data provide new insights into the involvement of MT-3 in a multiprotein complex, which will be used to understand the biological activity of MT-3 and its role in neurological disease.

Role of Zinc Metallothionein-3 (ZnMt3) in Epidermal Growth Factor (EGF)-induced c-Abl Protein Activation and Actin Polymerization in Cultured Astrocytes

“…3), we examined whether Mt3 binding to actin (23,30) was responsible for this change. To accomplish this, we transfected WT astrocytes with GFP-Mt3 or GFP-C1 plasmid DNA (negative control) and incubated cells with phalloidin to stain for F-actin.…”

“…Of four isoforms (Mt1-4), metallothionein-3 (Mt3) is most prominently expressed in the central nervous system (16,17); it is also expressed in cultured astrocytes (18 -22). Mt3 binds many different proteins, including actin (23,24). However, the biological function of Mt3, especially regarding the actin-Mt3 binding, has not been characterized.…”

Induction of metallothionein in mouse cerebellum and cerebrum with low-dose thimerosal injection

“…In the brain, it is known that MT-1 and MT-2 exist in glia and astrocyte cells, and MT-3 in neurons (Nishimura et al 1992;Coyle et al 2002;Ghazi et al 2006). MT-1 and MT-2 are induced simultaneously in the brain when various stimuli, including mercury vapor and methyl mercury, affect the brain (LeyshonSørland et al 1994;Yasutake et al 2003Yasutake et al , 2004, and they probably act as detoxification substances (Penkowa and Hidalgo 2000;Penkowa et al 2006).…”

Metallothionein-3 (MT-3) in the Human Adrenal Cortex and its Disorders

“…However, details on MT-3 expression within the NA cortical zonation or other types of adrenocortical adenoma were not evaluated. Although MT-1/2 have been previously reported in the adrenal cortex [16], their function and transcriptional regulation is postulated to be different from that of MT-3, which is not induced by heavy metals like the former, and is suggested to be more involved in cell growth inhibition and metabolism control than the MT-1/2 isoforms [17][18][19][20].…”