Zinc affects the proteolytic stability of Apolipoprotein E in an isoform-dependent way

Highlights

• Zinc destabilizes ApoE in the presence of proteases in an order of ApoE4 > ApoE2 and ApoE3.

• Plasma ApoE levels follow an order of ApoE2 > ApoE3 > ApoE4/

• Plasma ApoE levels are not affected by metals.

Abstract

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4 > E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD.

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.