Janet R. Kumita

The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures

Clusterin is an extracellular chaperone present in all disease‐associated extracellular amyloid deposits, but its roles in amyloid formation and protein deposition in vivo are poorly understood. The current study initially aimed to characterize the effects of clusterin on amyloid formation in vitro by a panel of eight protein substrates. Two of the substrates (Alzheimers beta peptide and a PI3‐SH3 domain) were then used in further experiments to examine the effects of clusterin on amyloid cytotoxicity and to probe the mechanism of clusterin action. We show that clusterin exerts potent effects on amyloid formation, the nature and extent of which vary greatly with the clusterin: substrate ratio, and provide evidence that these effects are exerted via interactions with prefibrillar species that share common structural features. Proamyloido‐genic effects of clusterin appear to be restricted to conditions in which the substrate protein is present at a very large molar excess;under these same conditions, clusterin coincorporates with substrate protein into insoluble aggregates. However, when clusterin is present at much higher but still substoichiometric levels (e.g., a molar ratio of clusterin:substrate = 1:10), it potently inhibits amyloid formation and provides substantial cytoprotection. These findings suggest that clus‐terin is an important element in the control of extracellular protein misfolding.—Yerbury, J. J., Poon, S., Meehan, S., Thompson, B., Kumita, J. R., Dobson, C. M., Wilson, M. R. The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures. FASEB J. 21, 2312–2322 (2007)

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Significance Amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease, is associated with mutation and misfolding of the Cu/Zn superoxide dismutase (SOD1) protein. Prior studies found that mutant misfolded SOD1 can convert wild-type (WT) SOD1 to a misfolded form inside living cells in a prion-like fashion. We now report that misfolded WT SOD1 can be transmitted from cell to cell, and that propagated protein misfolding can be perpetuated. Misfolded SOD1 transmission between cells can be mediated through release and uptake of protein aggregates or via small membrane-bounded transport vesicles called exosomes. These mechanisms may help explain why sporadic ALS, without a known genetic cause, can spread systematically from region to region in a progressive manner. Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Molecular determinants of the aggregation behavior of α‐ and β‐synuclein

α‐ and β‐synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinsons disease while the second appears to have no relationship to any such disorders. The aggregation behavior of α‐ and β‐synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11‐residue sequence of mainly hydrophobic residues from the non‐amyloid‐β‐component (NAC) region of α‐synuclein into β‐synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of β‐synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation‐prone region of the NAC region of α‐synuclein. This conclusion provides new insights into the role of α‐synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.

α2-Macroglobulin and Haptoglobin Suppress Amyloid Formation by Interacting with Prefibrillar Protein Species

α2-Macroglobulin (α2M) and haptoglobin (Hp) are both abundant secreted glycoproteins that are best known for their protease trapping and hemoglobin binding activities, respectively. Like the small heat shock proteins, both these glycoproteins have in common the ability to protect a range of proteins from stress-induced amorphous aggregation and have been described as extracellular chaperones. Using an array of biophysical techniques, this study establishes that in vitro at substoichiometric levels and under physiological conditions α2M and Hp both inhibit the formation of amyloid fibrils from a range of proteins. We also provide evidence that both α2M and Hp interact with prefibrillar species to maintain the solubility of amyloidogenic proteins. These findings suggest that both α2M and Hp are likely to play an important role in controlling the inappropriate aggregation of proteins in the extracellular environment.

Engineering a Camelid Antibody Fragment That Binds to the Active Site of Human Lysozyme and Inhibits Its Conversion into Amyloid Fibrils

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.

Towards Multiparametric Fluorescent Imaging of Amyloid Formation: Studies of a YFP Model of α-Synuclein Aggregation

Misfolding and aggregation of proteins are characteristics of a range of increasingly prevalent neurodegenerative disorders including Alzheimers and Parkinsons diseases. In Parkinsons disease and several closely related syndromes, the protein alpha-synuclein (AS) aggregates and forms amyloid-like deposits in specific regions of the brain. Fluorescence microscopy using fluorescent proteins, for instance the yellow fluorescent protein (YFP), is the method of choice to image molecular events such as protein aggregation in living organisms. The presence of a bulky fluorescent protein tag, however, may potentially affect significantly the properties of the protein of interest; for AS in particular, its relative small size and, as an intrinsically unfolded protein, its lack of defined secondary structure could challenge the usefulness of fluorescent-protein-based derivatives. Here, we subject a YFP fusion of AS to exhaustive studies in vitro designed to determine its potential as a means of probing amyloid formation in vivo. By employing a combination of biophysical and biochemical studies, we demonstrate that the conjugation of YFP does not significantly perturb the structure of AS in solution and find that the AS-YFP protein forms amyloid deposits in vitro that are essentially identical with those observed for wild-type AS, except that they are fluorescent. Of the several fluorescent properties of the YFP chimera that were assayed, we find that fluorescence anisotropy is a particularly useful parameter to follow the aggregation of AS-YFP, because of energy migration Förster resonance energy transfer (emFRET or homoFRET) between closely positioned YFP moieties occurring as a result of the high density of the fluorophore within the amyloid species. Fluorescence anisotropy imaging microscopy further demonstrates the ability of homoFRET to distinguish between soluble, pre-fibrillar aggregates and amyloid fibrils of AS-YFP. Our results validate the use of fluorescent protein chimeras of AS as representative models for studying protein aggregation and offer new opportunities for the investigation of amyloid aggregation in vivo using YFP-tagged proteins.

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild‐type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200‐ and 30‐fold lower, respectively, than that of wild‐type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post‐transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease‐associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native‐state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

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.

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)

Analysis of the Native Structure, Stability and Aggregation of Biotinylated Human Lysozyme

Fibril formation by mutational variants of human lysozyme is associated with a fatal form of hereditary non-neuropathic systemic amyloidosis. Defining the mechanistic details of lysozyme aggregation is of crucial importance for understanding the origin and progression of this disease and related misfolding conditions. In this study, we show that a biotin moiety can be introduced site-specifically at Lys33 of human lysozyme. We demonstrate, using biophysical techniques, that the structure and stability of the native-state of the protein are not detectably altered by this modification, and that the ability to form amyloid fibrils is unchanged. By taking advantage of biotin-avidin interactions, we show that super-resolution fluorescence microscopy can generate detailed images of the mature fibrils. This methodology can readily enable the introduction of additional probes into the protein, thereby providing the means through which to understand, in detail, the nature of the aggregation process of lysozyme and its variants under a variety of conditions.