Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.     

Effects of Hofmeister ions on the α-helical structure of proteins

The molecular conformation of proteins is sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. Here, we combine circular dichroism (CD), single-molecule Förster resonance energy transfer, and atomistic computer simulations to elucidate salt-specific effects on the structure of three peptides with large α-helical propensity. CD indicates a complex ion-specific destabilization of the α-helix that can be rationalized by using a single salt-free computer simulation in combination with the recently introduced scheme of ion-partitioning between nonpolar and polar peptide surfaces. Simulations including salt provide a molecular underpinning of this partitioning concept. Furthermore, our single-molecule Förster resonance energy transfer measurements reveal highly compressed peptide conformations in molar concentrations of NaClO₄ in contrast to strong swelling in the presence of GdmCl. The compacted states observed in the presence of NaClO₄ originate from a tight ion-backbone network that leads to a highly heterogeneous secondary structure distribution and an overall lower α-helical content that would be estimated from CD. Thus, NaClO₄ denatures by inducing a molten globule-like structure that seems completely off-pathway between a fully folded helix and a coil state.     

Elastase inhibition by the C-terminal domains of α-crystallin and small heat-shock protein

α-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of α-crystallin, αA and αB,also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both αA-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.     

Structural and mechanistic implications of metal binding in the small heat-shock protein αB-crystallin

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.     

Understanding integration of α-helical membrane proteins: the next steps

Integration of a protein into the endoplasmic reticulum (ER) membrane occurs through a series of multistep reactions that include targeting of ribosome-nascent polypeptide complexes to the ER, attachment of the ribosome to the protein translocation channel, lateral partitioning of α-helical transmembrane spans into the lipid bilayer, and folding of the lumenal, cytosolic and membrane-embedded domains of the protein. However, the molecular mechanisms and kinetics of these steps are still not entirely clear. To obtain a better understanding of the mechanism of membrane protein integration, we propose that it will be important to utilize in vivo experiments to examine the kinetics of membrane protein integration and in vitro experiments to characterize interactions between nascent membrane proteins, protein translocation factors and molecular chaperones.     

A survey of integral α-helical membrane proteins

Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families.     

γδ T cells lyse autologous and allogenic oesophageal tumours: involvement of heat-shock proteins in the tumour cell lysis

T cells expressing γδ receptors were isolated from the peripheral blood of oesophageal cancer patients and analysed for their potential to lyse tumour targets. Immunophenotyping by flow cytometry showed that the dominant population of γδ T cells expressed the Vγ9 and the Vδ2 T cell receptor, and a minor population expressed the Vδ1 receptor. Cytotoxicity assays revealed that activated γδ T cells lysed Daudi Burkitt's lymphoma and K562 cells. Lysis of autologous oesophageal tumours was higher than of allogenic tumours. Anti-hsp60 and anti-hsp70 mAb significantly inhibited the cytotoxicity of γδ T cells to both autologous and allogenic oesophageal tumours. Surface expression of hsp60 and hsp70 on oesophageal tumours and Daudi cells was demonstrated by flow cytometry. In conclusion, γδ T cells isolated from the peripheral blood of oesophageal cancer patients have the ability of kill oesophageal tumour cells. The lysis of tumour targets by the γδ T cells is brought about via recognition of heat-shock proteins expressed on the surface of tumour cells. γδ T cells isolated from the peripheral blood may have applications in adoptive immunotherapy of oesophageal cancer. Received: 15 April 1999 / Accepted: 16 September 1999     

The products of the “heat-shock” loci of Drosophila hydei

Antibody was raised against total Drosophila hydei embryonic cellular protein with a molecular weight between 65,000 and 70,000 dalton. This antiserum reacted with the 70,000 MW heat-shock peptide found, in ³⁵S labelled cell extracts of heat-shocked D. hydei tissue culture cells or salivary glands. — The antibody was coupled to Sepharose 4B and this material was used to absorb polysomes obtained from tissue culture cells incubated at 37° C in the presence of tritiated RNA precursors. The relative concentrations of various RNA species complementary to the heat-shock loci 2-32A, 2-36A, and 2-48C in either bound, non-bound, or total polysomal material was then determined by in situ hybridization. The RNA species complementary to locus 2-36A was found to be enriched in the bound polysomal material.     

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

The characterization of the whey proteins of guinea-pig milk: The isolation and properties of α-lactalbumin

1. The whey proteins of guinea-pig milk were examined by electrophoresis on paper, cellulose acetate, starch gel and polyacrylamide gel. 2. Two major proteins were detected, one of which was identified as blood serum albumin. 3. The major whey protein was isolated by CM-cellulose chromatography and on columns of Sephadex G-100. 4. The amino acid composition of the protein, taken in conjunction with its other properties, indicated that the major whey protein in guinea-pig milk is homologous with cow α-lactalbumin and that β-lactoglobulin is absent from guinea-pig milk. 5. Guinea-pig α-lactalbumin, which was obtained crystalline, had mol.wt. 15800, N-terminal lysine and C-terminal glutamine.     

Association of heat-shock proteins in various neurodegenerative disorders: is it a master key to open the therapeutic door?

A number of acute and chronic neurodegenerative disorders are caused due to misfolding and aggregation of many intra- and extracellular proteins. Protein misfolding and aggregation processes in cells are strongly regulated by cellular molecular chaperones known as heat-shock proteins (Hsps) that include Hsp60, Hsp70, Hsp40, and Hsp90. Recent studies have shown the evidences that Hsps are colocalized in protein aggregates in Alzheimer’s disease (AD), Parkinson’s disease (PD), Polyglutamine disease (PGD), Prion disease, and other neurodegenerative disorders. This fact indicates that Hsps might have attempted to prevent aggregate formation in cells and thus to suppress disease conditions. Experimental findings have already established in many cases that selective overexpression of Hsps like Hsp70 and Hsp40 prevented the disease progression in various animal models and cellular models. However, recently, various Hsp modulators like geldanamycin, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, and celastrol have shown to up-regulate the expression level of Hsp70 and Hsp40, which in turn triggers the solubilization of diseased protein aggregates. Hsps are, therefore, if appropriately selected, an attractive choice for therapeutic targeting in various kinds of neurodegeneration and hence are expected to have strong potential as therapeutic agents in suppressing or curing AD, PD, PGD, and other devastative neurodegenerative disorders. In the present review, we report the experimental findings that describe the implication of Hsps in the development of neurodegeneration and explore the possibility of how Hsps can be used directly or as a target by other agents to prevent various neurodegeneration through preventing aggregation process and thus reducing the toxicity of the oligomers based on the previous reports.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca²⁺ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA₃) and 5 mM CaCl₂ results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca²⁺ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca²⁺. The addition of Ca²⁺ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [³⁵S]methionine show that Ca²⁺ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca²⁺, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca²⁺ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca²⁺ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca²⁺ while Ba²⁺ is only 10% as effective. We conclude that Ca²⁺ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.     

Mammalian epidermal keratin Isolation and characterization of the α-helical proteins from newborn rat

Neutral buffer-insoluble proteins extracted from newborn rat epidermis with alkaline urea have been purified by chromatography on Sephadex G-150 columns run in the presence of sodium dodecyl sulfate. Two proteins with apparent molecular weights of 60 000 and 68 000, respectively have been isolated and characterized. Spectropolarimetric studies show both of them to be α-helical in contrast to the non-helical heavier and lighter species also solubilized with alkaline urea. The amino acid composition of the two proteins, their electrophoretic behavior and their immunological characteristics are essentially identical. Both proteins appear to be major constituents of rat epidermal tonofilaments.     

Solution NMR studies of polytopic α-helical membrane proteins

NMR spectroscopy has established itself as one of the main techniques for the structural study of integral membrane proteins. Remarkably, over the last few years, substantial progress has been achieved in the structure determination of increasingly complex polytopical α-helical membrane proteins, with their size approaching ～100kDa. Such advances are the result of significant improvements in NMR methodology, sample preparation and powerful selective isotope labelling schemes. We review the requirements facilitating such work based on the more recent solution NMR studies of α-helical proteins. While the majority of such studies still use detergent-solubilized proteins, alternative more native-like lipid-based media are emerging. Recent interaction, dynamics and conformational studies are discussed that cast a promising light on the future role of NMR in this important and exciting area.