Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.     

Cellular polyamines promote the aggregation of alpha-synuclein

The cellular polyamines putrescine, spermidine, and spermine accelerate the aggregation and fibrillization of alpha-synuclein, the major protein component of Lewy bodies associated with Parkinson's disease. Circular dichroism and fluorometric thioflavin T kinetic studies showed a transition of alpha-synuclein from unaggregated to highly aggregated states, characterized by lag and transition phases. In the presence of polyamines, both the lag and transition times were significantly shorter. All three polyamines accelerated the aggregation and fibrillization of alpha-synuclein to a degree that increased with the total charge, length, and concentration of the polyamine. Electron and scanning force microscopy of the reaction products after the lag phase revealed the presence of aggregated particles (protofibrils) and small fibrils. At the end of the transition phase, alpha-synuclein formed long fibrils in all cases, although some morphological variations were apparent. In the presence of polyamines, fibrils formed large networks leading ultimately to condensed aggregates. In the absence of polyamines, fibrils were mostly isolated. We conclude that the polyamines at physiological concentrations can modulate the propensity of alpha-synuclein to form fibrils and may hence play a role in the formation of cytosolic alpha-synuclein aggregates.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.     

Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling

Despite its importance in Parkinson's disease, a detailed understanding of the structure and mechanism of alpha-synuclein fibril formation remains elusive. In this study, we used site-directed spin labeling and electron paramagnetic resonance spectroscopy to study the structural features of monomeric and fibrillar alpha-synuclein. Our results indicate that monomeric alpha-synuclein, in solution, has a highly dynamic structure, in agreement with the notion that alpha-synuclein is a natively unfolded protein. In contrast, fibrillar aggregates of alpha-synuclein exhibit a distinct domain organization. Our data identify a highly ordered and specifically folded central core region of approximately 70 amino acids, whereas the N terminus is structurally more heterogeneous and the C terminus ( approximately 40 amino acids) is completely unfolded. Interestingly, the central core region of alpha-synuclein exhibits several features reminiscent of those observed in the core region of fibrillar Alzheimer's amyloid beta peptide, including an in-register parallel structure. Although the lengths of the respective core regions differ, fibrils from different amyloid proteins nevertheless appear to be able to take up highly similar, and possibly conserved, structures.     

Human Immunoglobulin Light Chains λ Form Amyloid Fibrils and Granular Aggregates in Solution

Myeloma nephropathy is a disorder characterized by deposition of monoclonal immunoglobulin light chains in the kidneys. The chains deposited form either amyloid fibrils or granular (amorphous) aggregates. Distinct molecular mechanisms leading to the formation of different aggregate types in kidney of patients with multiple myeloma are poorly understood. Here we describe the self-association kinetics of human monoclonal immunoglobulin light chains lambda (GRY) isolated from urine of a patient with multiple myeloma. Under physiological conditions, the isolated light chain exists predominantly in a form of covalent dimer with apparent molecular mass of 50.1 kD. Spectral probe binding, analytical gel filtration, Western blot analysis, and electron microscopy indicate that GRY dimer aggregation occurs via two different pathways producing either amyloid fibrils or amorphous aggregates depending on microenvironment. Incubation of GRY (25 microM) for 4-14 days at 37 degrees C in phosphate buffered saline (PBS), pH 7.0, or in PBS containing urea (0.8 M), pH 6.5, leads to amyloid fibril formation. Under electron microscopy, the fibrils show unbranched thread-like structures, approximately 60-80 x 1000 A in size, which can bind thioflavin T and Congo Red. GRY maintained in acetate buffer, pH 3.5, forms granular aggregates. The structure of GRY oligomers formed during the early stage of amyloid fibril formation (1-4 days) has been examined by means of protein cross-linking with homobifunctional reagents. These oligomers are predominantly trimers and tetramers.     

Monitoring the prevention of amyloid fibril formation by alpha-crystallin. Temperature dependence and the nature of the aggregating species

The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.     

Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Amyloid Protofibrils of Lysozyme Nucleate and Grow Via Oligomer Fusion

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.     

Unraveling the mysteries of protein folding and misfolding

This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for example, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.     

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed.     

Congo red populates partially unfolded states of an amyloidogenic protein to enhance aggregation and amyloid fibril formation

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.     

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.     

Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro

The aggregation of alpha-synuclein, involved in the pathogenesis of several neurodegenerative disorders such as Parkinson's disease, is enhanced in vitro by biogenic polyamines binding to the highly charged C-terminal region aa109-140. In this study, we investigated the influence of this region on the aggregation kinetics, monitored by thioflavin T binding and static light scattering, and morphology, assessed by electron microscopy, fluorescence microscopy, and turbidity, by comparing the effect of various solution conditions on the wild-type protein, the disease related mutants A53T and A30P, and two truncated variants, syn(1-108) and syn(1-124), lacking the complete or the C-terminal half of the polyamine binding site. In the presence of the intact C-terminus, aggregation was strongly retarded in physiological buffer. This inhibition of aggregation was overridden by (i) addition of spermine or MgCl(2) or lowering of pH, leading to strong charge shielding in the C-terminus or (ii) by truncation of aa125-140 or aa109-140. Addition of MgCl(2) or spermine or acidification were not effective in promoting aggregation of syn(1-108). The impact of the disease-related mutations on the aggregation kinetics was dependent on the solution conditions, with the aggregation propensity order A53T approximately wt > A30P at low ionic strength, but A53T > wt approximately A30P at high ionic strength, with exceedingly potent promotion of aggregation by the A53T mutation in the presence of spermine. In contrast to full-length alpha-synuclein aggregates, those formed from syn(1-108) did not exhibit a pronounced polymorphism. The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109-140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.     

Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation

The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy. When expressed from a multi-copy vector, TPO1 was located mainly on the plasma membrane, but with some localization on the vacuolar membrane. Polyamine transport by TPO1 was dependent on pH. Uptake of spermidine and spermine occurred at alkaline pH (pH 8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (pH 5.0). This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli. Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable with the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0. However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) exists mainly as spermidine (or spermine)-ribosome complex in cells. The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1. Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.     

Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer

Protein conformational changes that result in misfolding, aggregation and amyloid fibril formation are a common feature of many neurodegenerative disorders. Studies with beta-amyloid (Abeta), alpha-synuclein and other amyloid-forming proteins indicate that the assembly of misfolded protein conformers into fibrils is a complex process that may involve the population of metastable spherical and/or annular oligomeric assemblies. Here, we show by atomic force microscopy that a mutant huntingtin fragment with an expanded polyglutamine repeat forms spherical and annular oligomeric structures reminiscent of those formed by Abeta and alpha-synuclein. Notably, the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of neurodegeneration, modulate polyglutamine aggregation reactions by partitioning monomeric conformations and disfavoring the accretion of spherical and annular oligomers.     

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed.