The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV.

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.

Lipids in Amyloid-ß Processing, Aggregation, and Toxicity.

Aggregation of amyloid-beta (Aß) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aß has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aß from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aß generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aß or compete with it for the lipoprotein receptor binding, significantly influences Aß clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aß is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aß aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aß can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aß-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aß processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.

Monitoring the interaction between ß2-microglobulin and the molecular chaperone αB-crystallin by NMR and mass spectrometry: αB-crystallin dissociates ß2-microglobulin oligomers.

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein ß2-microglobulin (ß2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aß2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aß2m. From analysis of the NMR spectra collected at various R3Aß2m to αB-crystallin molar subunit ratios, it is concluded that the structured ß-sheet core and the apical loops of R3Aß2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type ß2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type ß2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated ß2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing ß2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.

A radish seed antifungal peptide with a high amyloid fibril-forming propensity.

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aß peptide.

The oligomerization of Aß peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aß and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aß peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aß fibrillation. The three histidine residues at positions 6, 13 and 14 of Aß(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils.

Amyloid fibril formation occurs from a wide range of peptides and proteins and is typically associated with a loss of protein function and/or a gain of toxic function, as the native structure of the protein undergoes major alteration to form a cross ß-sheet array. It is now well recognised that some amyloid fibrils have a biological function, which has led to increased interest in the potential that these so-called functional amyloids may either retain the function of the native protein, or gain function upon adopting a fibrillar structure. Herein, we investigate the molecular chaperone ability of α-crystallin, the predominant eye lens protein which is composed of two related subunits αA- and αB-crystallin, and its capacity to retain and even enhance its chaperone activity after forming aggregate structures under conditions of thermal and chemical stress. We demonstrate that both eye lens α-crystallin and αB-crystallin (which is also found extensively outside the lens) retain, to a significant degree, their molecular chaperone activity under conditions of structural change, including after formation into amyloid fibrils and amorphous aggregates. The results can be related directly to the effects of aging on the structure and chaperone function of α-crystallin in the eye lens, particularly its ability to prevent crystallin protein aggregation and hence lens opacification associated with cataract formation.

Molecular architecture of Aß fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aß plaque formation.

OBJECTIVES: The detailed structure of brain-derived Aß amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aß(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aß amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aß fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aß peptide fold is common to Aß fibrils.

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry.

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the 'lynchpin' for size expansion is highly specific. We demonstrate that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection.

Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants.

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Challenges and advances in the heterologous expression of cellulolytic enzymes: a review.

Second generation biofuel development is increasingly reliant on the recombinant expression of cellulases. Designing or identifying successful expression systems is thus of preeminent importance to industrial progress in the field. Recombinant production of cellulases has been performed using a wide range of expression systems in bacteria, yeasts and plants. In a number of these systems, particularly when using bacteria and plants, significant challenges have been experienced in expressing full-length proteins or proteins at high yield. Further difficulties have been encountered in designing recombinant systems for surface-display of cellulases and for use in consolidated bioprocessing in bacteria and yeast. For establishing cellulase expression in plants, various strategies are utilized to overcome problems, such as the auto-hydrolysis of developing plant cell walls. In this review, we investigate the major challenges, as well as the major advances made to date in the recombinant expression of cellulases across the commonly used bacterial, plant and yeast systems. We review some of the critical aspects to be considered for industrial-scale cellulase production.

Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.

Improvement of cellulase expression has the potential to change the nature of the biofuel industry. Increasing the economic feasibility of cellulase systems would significantly broaden the range of practicable biomass conversion, lowering the environmental impact of our civilisations' fuel needs. Cellulases are derived from certain fungi and bacteria, which are often difficult to culture on an industrial scale. Accordingly, methods to recombinantly express important cellulases and other glycosyl hydrolase (GH) enzymes are under serious investigation. Herein, we examine the latest developments in bacterial, yeast, plant, and fungal expression systems. We discuss current strategies for producing cellulases, and evaluate the benefits and drawbacks in yield, stability, and activity of enzymes from each system, and the overall progress in the field.

Amyloid fibrils from readily available sources: milk casein and lens crystallin proteins.

Amyloid fibrils are a highly ordered and robust aggregated form of protein structure in which the protein components are arranged in long fibrillar arrays comprised of ß-sheet. Because of these properties, along with their biocompatibility, amyloid fibrils have attracted much research attention as bionanomaterials, for example as template structures (in some cases following modification) that can be used as biosensors, encapsulators, and biomimetic materials. To use amyloid fibrils for such a range of applications will require them to be obtained relatively easily in large quantities. In this chapter, we describe methods for isolating crystallin and casein proteins from readily available sources that contain abundant protein, i.e., the eye lens and milk, respectively, and the subsequent conversion of these proteins into amyloid fibrils.

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support.

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

DHPC strongly affects the structure and oligomerization propensity of Alzheimer's Aß(1-40) peptide.

Alzheimer's disease (AD) is thought to depend on the deleterious action of amyloid fibrils or oligomers derived from ß-amyloid (Aß) peptide. Out of various known Aß alloforms, the 40-residue peptide Aß(1-40) occurs at highest concentrations inside the brains of AD patients. Its aggregation properties critically depend on lipids, and it was thus proposed that lipids could play a major role in AD. To better understand their possible effects on the structure of Aß and on the ability of this peptide to form potentially detrimental amyloid structures, we here analyze the interactions between Aß(1-40) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). DHPC has served, due to its controlled properties, as a major model system for studying general lipid properties. Here, we show that DHPC concentrations of 8 mM or higher exert dramatic effects on the conformation of soluble Aß(1-40) peptide and induce the formation of ß-sheet structure at high levels. By contrast, we find that DHPC concentrations well below the critical micelle concentration present no discernible effect on the conformation of soluble Aß, although they substantially affect the peptide's oligomerization and fibrillation kinetics. These data imply that subtle lipid-peptide interactions suffice in controlling the overall aggregation properties and drastically accelerate, or delay, the fibrillation kinetics of Aß peptide in near-physiological buffer solutions.