Role of protein aggregate structure on the strength and underwater performance of barnacle-inspired adhesives.

Nature employs protein aggregates when strong materials are needed to adhere surfaces in extreme environments, allowing organisms to survive conditions ranging from harsh intertidal coasts to open oceans. Amyloids and amyloid-like materials are prevalent and amongst the most densely bonded aggregate structures, though how they contribute to wet adhesion is not well understood. In this work, waterborne protein solutions of individual whey proteins are cured in place using varied temperature to produce model adhesives enriched in amyloid or non-amyloid aggregates. Dry adhesive strengths range from 0.2-1.5 MPa, while wet adhesive strengths range from 0-0.5 MPa across the tested proteins and processing conditions, highlighting that both proper protein selection and controlled aggregation extent are necessary for successful underwater performance. For bovine serum albumin, the amyloid-enriched adhesive was able to retain ca. 500 kPa bond strength underwater throughout extended immersion and thermal degradation testing, while the non-amyloid adhesive weakened by up to 80%. As freestanding gels, higher temperature processing improved underwater stability for all the protein materials, with amyloid-rich structures remaining mostly water-insoluble after 30 days submerged in water. Protein-based adhesives with a controlled aggregate structure shed light on the ability of amyloid-containing materials to remain adhered underwater, a necessary trait for the survival of many organisms.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Preparation protocols of aß(1-40) promote the formation of polymorphic aggregates and altered interactions with lipid bilayers.

The appearance of neuritic amyloid plaques comprised of ß-amyloid peptide (Aß) in the brain is a predominant feature in Alzheimer's disease (AD). In the aggregation process, Aß samples a variety of potentially toxic aggregate species, ranging from small oligomers to fibrils. Aß has the ability to form a variety of morphologically distinct and stable amyloid fibrils. Commonly termed polymorphs, such distinct aggregate species may play a role in variations of AD pathology. It has been well documented that polymorphic aggregates of Aß can be produced by changes in the chemical environment and peptide preparations. As Aß and several of its aggregated forms are known to interact directly with lipid membranes and this interaction may play a role in a variety of potential toxic mechanisms associated with AD, we determine how different Aß(1-40) preparation protocols that lead to distinct polymorphic fibril aggregates influence the interaction of Aß(1-40) with model lipid membranes. Using three distinct protocols for preparing Aß(1-40), the aggregate species formed in the absence and presence of a lipid bilayers were investigated using a variety of scanning probe microscopy techniques. The three preparations of Aß(1-40) promoted distinct oligomeric and fibrillar aggregates in the absence of bilayers that formed at different rates. Despite these differences in aggregation properties, all Aß(1-40) preparations were able to disrupt supported total brain lipid extract bilayers, altering the bilayer's morphological and mechanical properties.

Amyloid-forming proteins alter the local mechanical properties of lipid membranes.

A diverse number of diseases, including Alzheimer's disease, Huntington's disease, and type 2 diabetes, are characterized by the formation of fibrillar protein aggregates termed amyloids. The precise mechanism by which aggregates are toxic remains unclear; however, these proteins have been shown to interact strongly with lipid membranes. We investigated morphological and mechanical changes in model lipid bilayers exposed to amyloid-forming proteins by reconstructing the tapping forces associated with atomic force microscopy (AFM) imaging in solution. Tip/sample tapping forces contain information regarding mechanical properties of surfaces. Interpretation of the mechanical changes in the bilayers was aided by numerical simulations of the entire AFM experiment. Amyloid-forming proteins disrupted distinct regions of the bilayer morphology, and these regions were associated with decreased Young's modulus and adhesive properties. These changes in bilayer mechanical properties upon exposure to amyloid-forming proteins may represent a common mechanism leading to membrane dysfunction in amyloid diseases.

High-Throughput Screening of Heterologous Functional Amyloids Using Escherichia coli.

Escherichia coli remains one of the most widely used workhorse microorganisms for the expression of heterologous proteins. The large number of cloning vectors and mutant host strains available for E. coli yields an impressively wide array of folded globular proteins in the laboratory. However, applying modern functional screening approaches to interrogate insoluble protein aggregates such as amyloids requires the use of nonstandard expression pathways. In this chapter, we detail the use of the curli export pathway in E. coli to express a library of gene fragments and variants of a functional amyloid protein to screen sequence traits responsible for aggregation and the formation of nanoscale materials.

Structural Mimicry Drives HIV-1 Rev-Mediated HERV-K Expression.

Expression of the Human Endogenous Retrovirus Type K (HERV-K), the youngest and most active HERV, has been associated with various cancers and neurodegenerative diseases. As in all retroviruses, a fraction of HERV-K transcripts is exported from the nucleus in unspliced or incompletely spliced forms to serve as templates for translation of viral proteins. In a fraction of HERV-K loci (Type 2 proviruses), nuclear export of the unspliced HERV-K mRNA appears to be mediated by a cis-acting signal on the mRNA, the RcRE, and the protein Rec-these are analogous to the RRE-Rev system in HIV-1. Interestingly, the HIV-1 Rev protein is able to mediate the nuclear export of the HERV-K RcRE, contributing to elevated HERV-K expression in HIV-infected patients. We aimed to understand the structural basis for HIV Rev-HERV-K RcRE recognition. We examined the conformation of the RcRE RNA in solution using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the 433-nt long RcRE can assume folded or extended conformations as observed by AFM. SAXS analysis of a truncated RcRE variant revealed an "A"-shaped topological structure similar to the one previously reported for the HIV-1 RRE. The effect of the overall topology was examined using several deletion variants. SAXS and biochemical analyses demonstrated that the "A" shape is necessary for efficient Rev-RcRE complex formation in vitro and nuclear export activity in cell culture. The findings provide insight into the mechanism of HERV-K expression and a structural explanation for HIV-1 Rev-mediated expression of HERV-K in HIV-infected patients. IMPORTANCE: Expression of the human endogenous retrovirus type K (HERV-K) has been associated with various cancers and autoimmune diseases. Nuclear export of both HIV-1 and HERV-K mRNAs is dependent on the interaction between a small viral protein (Rev in HIV-1 and Rec in HERV-K) and a region on the mRNA (RRE in HIV-1 and RcRE in HERV-K). HIV-1 Rev is able to mediate the nuclear export of RcRE-containing HERV-K mRNAs, which contributes to elevated production of HERV-K proteins in HIV-infected patients. We report the solution conformation of the RcRE RNA-the first three-dimensional topological structure for a HERV molecule-and find that the RcRE resembles the HIV-1 nuclear export signal, RRE. The finding reveals the structural basis for the increased HERV-K expression observed in HIV-infected patients. Elevated HERV expression, mediated by HIV infection or other stressors, can have various HERV-related biological consequences. The findings provide structural insight for regulation of HERV-K expression.

Colorimetric Detection of Mutant ß-Amyloid(1-40) Membrane-Active Aggregation with Biosensing Vesicles.

Alzheimer's disease (AD) is a protein misfolding disease commonly characterized by neuritic amyloid plaques and proteinaceous fibrillar aggregate deposits composed of ß-amyloid (Aß) aggregates. The dynamic aggregation of Aß forms toxic, nanoscale aggregate species which proceed from oligomers to fibrils. Currently, there is need for rapid and direct detection of Aß peptide aggregation and interaction with lipid membranes, as detecting an interaction with various lipid environments will provide insights to better understand how interactions may modulate membrane function on cellular surfaces, leading to the progression of AD. The goal of this study was to utilize a colorimetric, biomimetic, vesicle-binding assay as a biosensor to detect and investigate the occurrence of neurodegenerative disease-associated protein aggregation and interaction with lipid membranes. Lipid/polydiacetylene (PDA) vesicles were exposed to monomeric preparations of Wild Type Aß(1-40) or point mutations in Aß with amino acid substitutions that are commonly associated with familial AD (E22G Arctic, E22Q Dutch, A21G Flemish, D23N Iowa, and E22K Italian). We investigated how these substitutions affect Aß(1-40) aggregation and interaction with lipid vesicles designed to mimic biological membranes. Time-resolved colorimetric measurements were obtained and reveal that exposure to lipid/PDA vesicle biosensors results in the direct detection of mutant Aß(1-40) peptide-lipid interaction events. Aß(1-40) peptide aggregate membrane activity varies among Aß peptide variants and lipid composition. In addition, we used atomic force microscopy and Thioflavin T fluorescence assays to distinguish the stages of Aß(1-40) aggregate formation, morphology, and membrane activity. These studies provide a simple means of aggregate detection and insight into the role of cellular surfaces in the mechanism of AD aggregation.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration.

There are a vast number of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes.

Specific domains of Aß facilitate aggregation on and association with lipid bilayers.

A hallmark of Alzheimer's disease, a late-onset neurodegenerative disease, is the deposition of neuritic amyloid plaques composed of aggregated forms of the ß-amyloid peptide (Aß). Aß forms a variety of nanoscale, toxic aggregate species ranging from small oligomers to fibrils. Aß and many of its aggregate forms strongly interact with lipid membranes, which may represent an important step in several toxic mechanisms. Understanding the role that specific regions of Aß play in regulating its aggregation and interaction with lipid membranes may provide insights into the fundamental interaction between Aß and cellular surfaces. We investigated the interaction and aggregation of several Aß fragments (Aß1-11, Aß1-28, Aß10-26, Aß12-24, Aß16-22, Aß22-35, and Aß1-40) in the presence of supported model total brain lipid extract (TBLE) bilayers. These fragments represent a variety of chemically unique domains within Aß, that is, the extracellular domain, the central hydrophobic core, and the transmembrane domain. Using scanning probe techniques, we elucidated aggregate morphologies for these different Aß fragments in free solution and in the presence of TBLE bilayers. These fragments formed a variety of oligomeric and fibrillar aggregates under free solution conditions. Exposure to TBLE bilayers resulted in distinct aggregate morphologies compared to free solution and changes in bilayer stability dependent on the Aß sequence. Aß10-26, Aß16-22, Aß22-35, and Aß1-40 aggregated into a variety of distinct fibrillar aggregates and disrupted the bilayer structure, resulting in altered mechanical properties of the bilayer. Aß1-11, Aß1-28, and Aß12-24 had minimal interaction with lipid membranes, forming only sparse oligomers.

Point mutations in Aß result in the formation of distinct polymorphic aggregates in the presence of lipid bilayers.

A hallmark of Alzheimer's disease (AD) is the rearrangement of the ß-amyloid (Aß) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aß toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aß near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aß aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aß, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aß, which alter peptide charge and hydrophobic character, influence interactions between Aß and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.

Point mutations in Aß induce polymorphic aggregates at liquid/solid interfaces.

A pathological hallmark of Alzheimer's disease (AD), a late onset neurodegenerative disease, is the development of neuritic amyloid plaques, composed predominantly of aggregates of the ß-amyloid (Aß) peptide. It has been demonstrated that Aß can aggregate into a variety of polymorphic aggregate structures under different chemical environments, and a potentially important environmental factor in dictating aggregate structure is the presence of surfaces. There are also several mutations clustered around the central hydrophobic core of Aß (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology. The goal of this study was to determine how these mutations influence the morphology of Aß aggregates under free solution conditions and at an anionic surface/liquid interface. While the rate of formation of specific aggregates was altered by mutations in Aß under free solution conditions, the respective aggregate morphologies were similar. However, aggregation occurring directly on a negatively charged mica surface resulted in distinct aggregate morphologies formed by different mutant forms of Aß. These studies provide insight into the potential role anionic surfaces play in dictating the formation of Aß polymorphic aggregate structures.