Zinc affects the proteolytic stability of Apolipoprotein E in an isoform-dependent way
Introduction
Apolipoprotein E protein (ApoE) is a 34 kDa polymorphic glycoprotein existing in the brain and periphery. Three ApoE isoforms—ApoE2, ApoE3 and ApoE4 are encoded by the same Apolipoprotein E gene (APOE) with three alleles (APOE2, APOE3 and APOE4) (Mahley, 1988). APOE4, which accounts for 10%–15% of the APOE gene pool in the population, has been identified as the strongest genetic risk factor for late onset Alzheimer's disease (LOAD), increasing the risk level by three times in heterozygous individuals and by twelve times in homozygous individuals (Mahley, 1988, Corder et al., 1993). The least frequent APOE2 allele (5–10%) appears to protect against AD (Mahley, 1988, Corder et al., 1994), while the most frequent APOE3 allele (70–80%) represents an intermediate risk for disease development (Huang, 2006).
ApoE contains two structural domains by a protease sensitive hinge region: a 22-kDa amino-terminal domain (NT, residue 1–191) that has a four-helix structure and contains the low-density lipoprotein receptor-binding region (residue 136–150) and a 10 kDa carboxyl-terminal domain (CT, residue 216–299) existing as an α-helical bundle and containing the major lipid-binding region (residue 244–272) and probably amyloid-β (Frieden and Garai, 2012). ApoE isoforms differ at two amino acid positions, 112 and 158. ApoE2 has cysteine-112 and cysteine-158, whereas ApoE4 has arginine at both positions and ApoE3 has cysteine-112 and arginine-158 (Mahley and Huang, 2006). Since cysteine is a strong ligand for zinc and arginine is not (Karlin and Zhu, 1997), it is speculated that ApoE2 has the highest affinity for zinc while ApoE4 has the lowest. In addition, this cysteine/arginine difference has a dramatic influence on two critical properties of ApoE: protein stability and domain interactions (Huang, 2010). Denaturation studies have demonstrated that ApoE4 is the least stable isoform, whereas ApoE2 is most stable (Morrow et al., 2000, Morrow et al., 2002). The ApoE4 instability leads to the formation of an ensemble of loosely folded structures referred to as a molten globule state while ApoE2 is most resistant to this, and this increases ApoE4's susceptibility to proteolysis compared to ApoE2 and ApoE3 (Morrow et al., 2002). These effects likely contribute to the interaction of ApoE4 in AD.
Given the strong association between ApoE genotype and the risk of developing AD, numerous studies have investigated whether levels of ApoE are altered in AD patients. Total plasma ApoE levels have been reported to be significantly lower in AD patients, irrespective of APOE genotypes and APOE4 carriers exhibited the largest decrease in total plasma ApoE levels (Gupta et al., 2011, Wang et al., 2014). Likewise, several studies have also reported genotype-dependent variability in ApoE levels in the brain with the lowest concentrations found in APOE4 carriers (Hudry et al., 2013, Riddell et al., 2008, Ulrich et al., 2013). Several enzymes, such as thrombin and chymotrypsin-like serine protease, have been suggested to mediate ApoE proteolysis to decrease ApoE levels and generate neurotoxic fragments in the brain (Elliott et al., 2011, Marques et al., 2004, Harris et al., 2003, Tamboli et al., 2014). ApoE fragments are found in AD brains associated with amyloid plaques and APOE4 carriers have the most ApoE fragments. Differential proteolysis of ApoE isoforms and toxicity exerted by its fragments is believed to play an important role in AD pathogenesis (Huang and Mucke, 2012). The potential role/interaction of metal ion homeostasis in this toxicity of ApoE has been recently reviewed (Xu et al., 2014).
Briefly, alterations in zinc homeostasis have been suggested to be a key factor in the progression to AD (Barnham and Bush, 2014). Zinc is a potent inducer of Aβ aggregation and is concentrated within the extracellular amyloid plaques (Bush and Tanzi, 2002). Zinc deficiency has been demonstrated in older healthy individuals, with serum zinc levels even lower in AD patients (Baum et al., 2010). Of relevance to this study, it has also been reported that zinc is significantly increased in the serum of APOE4 positive AD patients, suggesting the presence of APOE4 alone may increase zinc levels (Gonzalez et al., 1999). Intriguingly, ApoE has an affinity for zinc (Miyata and Smith, 1996) and their correlation in the development of AD is gaining increasing attention (Xu et al., 2014). The metal sequestration properties of ApoE might present metals to Aβ peptides, leading to amyloid deposition or it might account for the antioxidant function of ApoE in AD development (Miyata and Smith, 1996). In this study we investigated the influence of zinc on the proteolytic stability of ApoE. We found that zinc is able to promote ApoE degradation by proteases in an isoform-dependent way (E4 > E2 and E3), which might contribute to AD pathogenesis.
Section snippets
ApoE preparation
Recombinant ApoE2, ApoE3 and ApoE4 (Escherichia coli, r-ApoE) were purchased from Sigma-Aldrich. The peptides were dissolved in Tris-buffered saline (TBS, 20 mM Tris–HCl and 150 mM NaCl, pH = 7.4) and stored at − 20 °C until further use. The final concentration of ApoE in our experiment was 40 mg/L, which represents the physiological concentration of ApoE in human plasma (Wang et al., 2014). In addition, lipidated ApoE isoforms were obtained from our collaborators: Prof. Ralph Martins and Ian Martins
Zinc and copper do not affect the stability or immunoreactivity of r-ApoE isoforms
Non-lipidated r-ApoEs were incubated with zinc and copper (10 and 100 μM) at 37 °C for 4 h (Fig. 1A). The incubation itself did not affect ApoE levels as there was no difference in full-length ApoE (34 kDa) levels between the “no incubation” (NI) and “TBS incubation” groups. We also observed that r-ApoEs levels did not change after zinc or copper treatment and 300 μM of chelators (TPEN, BCS, DPEN and EDTA) had no influence on the r-ApoEs levels. Lipidated r-ApoE samples were treated similarly and
Discussion
Zinc has been demonstrated to play an important role in the pathogenesis of AD (Barnham and Bush, 2014), facilitating Aβ aggregation (Bush and Tanzi, 2002, Huang et al., 2000) as well as tau phosphorylation and neurofibrillary tangle (NFTs) formation (Xiong et al., 2013). The precise mechanism by which zinc contributes to disease, however, remains unknown. Our study shows that zinc influences the proteolytic degradation of ApoE, which is the most important genetic risk factor for the sporadic
Conclusions
By investigating ApoE proteolytic stability, we present evidence supporting an interaction between zinc and ApoE. We propose that the ability of zinc to promote ApoE proteolysis may contribute to the onset and/or progression of AD in the ageing brain. Furthermore, the isoform-specific effects of zinc may account for the higher risk for AD in APOE4 carriers. Further studies on detailed mechanism of the interactions still need to be clarified.
The following are the supplementary related to this
Conflict of interest
None declared.
Acknowledgments
PAA is supported by an ARC Future Fellowship. In addition, the Florey Institute of Neuroscience and Mental Health acknowledge the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant. He Xu is supported by a University of Melbourne Scholarship. We wish to thank the donors of blood samples, who were participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) (http://www.aibl.csiro.au/).
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2022, Neurobiology of DiseaseCitation Excerpt :The 22 kDa fragment generated by thrombin from ApoE4 showed higher toxicity to neurons than those from ApoE3, which are also found in Aβ plaques and NFTs in post-mortem human tissue (Arai et al., 2006; Marques et al., 1996; Tolar et al., 1997). Our previous work has demonstrated that zinc can promote the proteolysis of synthetic ApoE in the order ApoE4>ApoE2=ApoE3, resulting in more ApoE4 fragments that are toxic to neurons (Xu et al., 2015). Thus, the isoform-specific effects of ApoE on AD pathology could be due, at least in part, to the elevated brain zinc.
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2022, International Journal of Biochemistry and Cell BiologyCitation Excerpt :The Cys residues in the E2 and E3 alleles allow for metals to bind, thus limiting their availability to interact with Aβ. Zinc also shows an isoform-dependent effect on APOE proteolysis (Xu et al., 2015). Xu and colleagues demonstrated that Zn caused an increase in proteolytic activity of APOE4 through the enzymes thrombin and chymotrypsin, more so than the E2 and E3 isoforms.
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2021, Journal of Biological ChemistryCitation Excerpt :There are several reports of zinc interacting also with the other major proteins implicated in AD. Zinc is reported to increase presenilin 1 expression (108) and to affect the stability of apolipoprotein E (ApoE), particularly ApoE4 (109). Conversely, presenilin 1 and ApoE expression have been reported to play essential roles in maintaining cellular and neuronal zinc trafficking (108, 110).
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2018, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :The association between APOE and zinc appears to be at multiple levels. APOE isoforms bind zinc directly [84], and this binding can increase their stability [85]. In addition, it is believed that zinc can increase cellular APOE levels by directly affecting transcription [86] and/or secretion [87].
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Contributed equally.