Amy R. Wyatt

Quality control of protein folding in extracellular space.

The pathologies of many serious human diseases are thought to develop from the effects of intra‐ or extracellular aggregates of non‐native proteins. Inside cells, chaperone and protease systems regulate protein folding; however, little is known about any corresponding mechanisms that operate extracellularly. The identification of these mechanisms is important for the development of new disease therapies. This review briefly discusses the consequences of protein misfolding, the intracellular mechanisms that control folding and the potential corresponding extracellular control processes. Finally, a new speculative model is described, which proposes that newly discovered extracellular chaperones bind to exposed regions of hydrophobicity on non‐native, extracellular proteins to target them for receptor‐mediated endocytosis and intracellular, lysosomal degradation.

Clusterin facilitates in vivo clearance of extracellular misfolded proteins.

The extracellular deposition of misfolded proteins is a characteristic of many debilitating age-related disorders. However, little is known about the specific mechanisms that act to suppress this process in vivo. Clusterin (CLU) is an extracellular chaperone that forms stable and soluble complexes with misfolded client proteins. Here we explore the fate of complexes formed between CLU and misfolded proteins both in vitro and in a living organism. We show that proteins injected into rats are cleared more rapidly from circulation when complexed with CLU as a result of their more efficient localization to the liver and that this clearance is delayed by pre-injection with the scavenger receptor inhibitor fucoidan. The CLU–client complexes were found to bind preferentially, in a fucoidan-inhibitable manner, to human peripheral blood monocytes and isolated rat hepatocytes and in the latter cell type were internalized and targeted to lysosomes for degradation. The data suggest, therefore, that CLU plays a key role in an extracellular proteostasis system that recognizes, keeps soluble, and then rapidly mediates the disposal of misfolded proteins.

Extracellular Chaperones and Proteostasis

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the worlds most debilitating, costly, and intractable diseases.

Structural Characterization of Clusterin-Chaperone Client Protein Complexes

Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU ≥ 4 × 107 Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57 ± 18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06 ± 6.87 nm and 52.61 ± 7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins.

Roles of Extracellular Chaperones in Amyloidosis

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimers disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

Hypochlorite-induced structural modifications enhance the chaperone activity of human α2-macroglobulin.

Significance Hypochlorite is a powerful oxidant that is generated within the body by activated innate immune cells. When hypochlorite is produced, the host organism sustains collateral damage, particularly to proteins, and the accumulation of damaged (misfolded) proteins is a hallmark of inflammatory processes (e.g., in Alzheimer’s disease, atherosclerosis, and arthritis). In the present study, we show that the chaperone activity of human α2-macroglobulin, a highly abundant secreted protein, is dramatically increased by hypochlorite-induced structural modifications. The data support the conclusion that α2-macroglobulin is a unique component of the innate immune system that is posttranslationally regulated by hypochlorite to facilitate the clearance of potentially pathogenic misfolded proteins. Hypochlorite, an oxidant generated in vivo by the innate immune system, kills invading pathogens largely by inducing the misfolding of microbial proteins. Concomitantly, the nonspecific activity of hypochlorite also damages host proteins, and the accumulation of damaged (misfolded) proteins is implicated in the pathology of a variety of debilitating human disorders (e.g., Alzheimer’s disease, atherosclerosis, and arthritis). It is well-known that cells respond to oxidative stress by up-regulating proteostasis machinery, but the direct activation of mammalian chaperones by hypochlorite has not, to our knowledge, been previously reported. In this study, we show that hypochlorite-induced modifications of human α2-macroglobulin (α2M) markedly increase its chaperone activity by generating species, particularly dimers formed by dissociation of the native tetramer, which have enhanced surface hydrophobicity. Moreover, dimeric α2M is generated in whole-blood plasma in the presence of physiologically relevant amounts of hypochlorite. The chaperone activity of hypochlorite-modified α2M involves the formation of stable soluble complexes with misfolded client proteins, including heat-denatured enzymes, oxidized fibrinogen, oxidized LDL, and native or oxidized amyloid β-peptide (Aβ1–42). Here, we show that hypochlorite-modified α2M delivers its misfolded cargo to lipoprotein receptors on macrophages and reduces Aβ1–42 neurotoxicity. Our results support the conclusion that α2M is a specialized chaperone that prevents the extracellular accumulation of misfolded and potentially pathogenic proteins, particularly during innate immune system activity.

Identification of Human Plasma Proteins as Major Clients for the Extracellular Chaperone Clusterin

Clusterin (CLU) is an extracellular chaperone that is likely to play an important role in protein folding quality control. This study identified three deposition disease-associated proteins as major plasma clients for clusterin by studying CLU-client complexes formed in response to physiologically relevant stress (shear stress, ∼36 dynes/cm2 at 37 °C). Analysis of plasma samples by size exclusion chromatography indicated that (i) relative to control plasma, stressed plasma contained proportionally more soluble protein species of high molecular weight, and (ii) high molecular weight species were far more abundant when proteins purified by anti-CLU immunoaffinity chromatography from stressed plasma were compared with those purified from control plasma. SDS-PAGE and Western blot analyses indicated that a variety of proteins co-purified with CLU from both stressed and control plasma; however, several proteins were uniquely present or much more abundant when plasma was stressed. These proteins were identified by mass spectrometry as ceruloplasmin, fibrinogen, and albumin. Immunodot blot analysis of size exclusion chromatography fractionated plasma suggested that CLU-client complexes generated in situ are very large and may reach ≥4 × 107 Da. Lastly, sandwich enzyme-linked immunosorbent assay detected complexes containing CLU and ceruloplasmin, fibrinogen, or albumin in stressed but not control plasma. We have previously proposed that CLU-client complexes serve as vehicles to dispose of damaged misfolded extracellular proteins in vivo via receptor-mediated endocytosis. A better understanding of these mechanisms is likely to ultimately lead to the identification of new therapies for extracellular protein deposition disorders.

Protease-activated alpha-2-macroglobulin can inhibit amyloid formation via two distinct mechanisms

Aβ1–42 and Aβ1–42 bind by fluorescence technology (View interaction) I59T lysozyme and I59T lysozyme bind by light scattering (View interaction) I59T lysozyme and I59T lysozyme bind by fluorescence technology (View interaction)Alpha‐lactalbumin and Alpha‐lactalbumin bind by fluorescence technology (View interaction) I59T lysozyme and I59T lysozyme bind by electron microscopy (View interaction) Aβ1–42 and Aβ1–42 bind by electron microscopy (View interaction)

PZP and PAI-2: Structurally-diverse, functionally similar pregnancy proteins?

Pregnancy zone protein (PZP) and plasminogen activator inhibitor type 2 (PAI-2) are two multifunctional proteins that are elevated in normal pregnancy and numerous other inflammatory states. Both proteins were originally identified as protease inhibitors, but current evidence supports the notion that they may also function as modulators of T-helper cells and/or extracellular chaperones. Exacerbated inflammation, fibrinolytic disturbances and misfolded proteins are all implicated in the pathology of preeclampsia, a leading cause of maternal and foetal mortality and morbidity. Notably, reduced levels of PZP or PAI-2 are associated with preeclampsia and clarification of their diverse functions in normal pregnancy could provide much needed insight regarding the pathogenesis of this disorder. Given that inflammation and protein misfolding underlie the pathology of a very large number of disorders, the contributions of PZP and PAI-2 to extracellular proteostasis and immunoregulation could be broad-reaching.

Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.