Preparation, Conjugation, and Stabilization of Amyloid-β Peptides
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Preparation, Conjugation, and Stabilization of Amyloid-β Peptides


Chapter 1 presents the development of an efficient method for the expression and purification of aggregation-prone amyloid-β (Aβ) peptides, including Aβ(M1-42), 15N-labeled Aβ(M1-42), and Aβ(M1-42) familial mutants. Aβ peptides are central to the pathogenesis of Alzheimer’s disease. Aβ peptides are highly aggregation-prone, making them challenging to prepare and purify. Advances in amyloid research rely on improved access to Aβ peptides. Chemical synthesis of Aβ can lead to impurities, such as amino acid deletion products, that are difficult to eliminate during purification. Expression of Aβ(1-42) peptide requires the generation of fusion protein and cleavage by protease to remove the N-terminal methionine group that originates from the translational start codon, which can make the preparation of Aβ costly and time-intensive.

In this chapter, I collaborated with fellow graduate student Stan Yoo to express Aβ(M1-42), a widely used form of Aβ with properties comparable to those of the native Aβ(1−42) peptide. Expression of Aβ(M1−42) is simple to execute and avoids the expensive and difficult enzymatic cleavage step associated with expression and isolation of Aβ(1−42). We then developed an efficient method to afford Aβ(M1-42) and 15N-labeled Aβ(M1-42) at around 19 mg per liter of bacterial culture with high purity using simple and inexpensive steps in three days. This method relies on the combination of protein biology tools such as bacterial expression and inclusion body solubilization, as well as peptide chemistry tools such as preparative HPLC purification of the solubilized inclusion bodies. This chapter also describes a simple method for the construction of recombinant plasmids and the preparation of Aβ peptides containing familial mutations. These methods may enable experiments that would otherwise be hindered by insufficient access to Aβ, such as NMR experiments with 15N-labeled Aβ. We anticipate that this method can be adjusted for the expression and purification of other amyloidogenic proteins.

Chapter 2 presents the preparation of Aβ peptide with an N‑terminal cysteine [Aβ(C1–42)], the development of tailored chemical reaction conditions for the conjugation of aggregation-prone Aβ(C1–42) peptide with fluorophores or biotin, and the biophysical studies of labeled Aβ peptides.

N-terminally functionalized Aβ peptides are important in amyloid and Alzheimer’s disease research. Site-specific labeling on the N-terminus of Aβ minimizes perturbation in the structure and function of the peptide, as the central and C-terminal regions of Aβ are more involved in fibril and oligomer formation. Although synthetic N-terminally functionalized Aβ peptides are commercially available, these peptides are expensive and limited to biotin and a few common fluorophores. Expressed Aβ peptides offer advantages over synthetic Aβ because they are free from amino acid deletions and epimeric contaminants, and they have been found to be more biological relevant because they aggregate more quickly and are more neurotoxic than synthetic Aβ.

The method of preparing Aβ(C1–42) relies on the hitherto unrecognized observation that the expression of the Aβ(MC1-42) gene yields the Aβ(C1–42) peptide, because the N-terminal methionine is endogenously excised by E. coli. This observation is significant, because it allows the preparation of a useful Aβ(C1–42) peptide without the additional N-terminal methionine that originates from the translational start codon. The Aβ(C1–42) peptide represents a minimal modification of native Aβ(1–42), and the addition of a single cysteine residue at the N-terminus enables labeling of the expressed Aβ with complete site specificity using widely available maleimide-based reagents.

The Aβ peptide is challenging to handle and label because of its strong propensity to aggregate. This chapter details the development of tailored chemical reaction conditions for handling and labeling of this aggregation-prone peptide. The labeling chemistry was optimized to be performed at pH 9 in HPLC fractions Aβ(C1–42), where Aβ remains mostly monomeric. Biophysical studies show that the labeled Aβ peptides behave like unlabeled Aβ and suggest that labeling of the N-terminus does not substantially alter the properties of the Aβ. This chapter also goes on to demonstrate the utility of the labeled Aβ peptides through fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. Ready access to labeled Aβ bearing fluorophores will advance amyloid and Alzheimer’s disease research by enabling experiments for investigating the pathogenic mechanism, transport, and clearance of Aβ, as well as for screening anti-Aβ antibodies.

Chapter 3 presents the introduction of intramolecular disulfide bonds to Aβ peptides that stabilizes Aβ into oligomeric state and the discovery of a disulfide-stapled peptide, AβC18C33, that forms homogeneous dimers and does not fibrilize. Aβ aggregates rapidly and exists as three different forms in equilibria: monomers, oligomers, and fibrils. Although Aβ fibrils are the most commonly found species in the brain tissue of patients with Alzheimer’s disease, soluble Aβ oligomers are the toxic contributors to neurodegeneration. Aβ oligomers are heterogenous and metastable: they can rapidly aggregate into more thermodynamically stable fibrils, which makes it challenging to isolate them and study their structures and biological properties. No high-resolution structures of Aβ oligomers have been reported thus far. Filling this gap is necessary for an enhanced understanding of the molecular basis of Alzheimer’s disease.

In this chapter, we aimed to generate stable, non-covalent Aβ oligomers by introducing intramolecular disulfide linkages to Aβ. The intramolecular linkages were designed to enforce a β-hairpin conformation, the key conformation that favors Aβ oligomer formation and disfavors fibrilization. We have designed, prepared, and studied a variety of mutant Aβ peptides containing intramolecular linkages. Among these mutant peptides, we discovered AβC18C33, an Aβ peptide containing a disulfide bond between positions 18 and 33, that forms stable, homogeneous dimers and does not fibrilize, as evidenced by the results of a series of biophysical studies.

The Aβ dimer has been proposed to be the basic building block of many larger Aβ oligomers and is thought to be one of the most neurotoxic and pathologically relevant species in Alzheimer’s disease. We thereby anticipate that the AβC18C33 peptide to serve as a stable, non-fibrilizing, and noncovalent Aβ dimer model for the exploration of the structure and pathogenesis of Aβ dimers, the generation of Aβ-specific antibodies, and the screening of Aβ-targeted drugs. Our laboratory is exploring ways to obtain high-resolution structures of this dimeric Aβ model peptide, using techniques including cryo-EM, NMR, and X-ray crystallography.

Chapter 4 presents the structure-based design of a cyclic peptide inhibitor towards SARS-CoV-2 using free molecular modeling and docking software and publicly available X-ray crystallographic structures. When the COVID-19 pandemic forced the temporary closure of our laboratory in March 2020, we began working on the structure-based drug design of inhibitors against the SARS-CoV-2 virus main protease. These efforts resulted in the design of a cyclic peptide inhibitor, UCI-1. Working with other group members, I published a tutorial paper based to describe the structure-based drug design process and teach others how to do it using the publicly available software UCSF Chimera and AutoDock Vina.SARS-CoV-2 is a highly infectious virus that causes COVID-19, a serious respiratory infection that has caused over 178 million infections and over 3.8 million deaths worldwide as of June 2021. Main protease is a crucial enzyme that SARS-CoV-2 utilizes for site-specifically cleaving the polyprotein that is translated from viral mRNA and generating mature proteins that are necessary for replication and infection. The essential role of main protease, as well as the success of HIV protease inhibitors in the treatment of AIDS, make main protease an attractive therapeutic target in the treatment of COVID-19.

This chapter begins with the analysis of the X-ray crystallographic structure of the main protease of the SARS coronavirus (SARS-CoV) bound to a peptide substrate. This chapter then describes the structural modification of the peptide substrate using the UCSF Chimera molecular modeling software to create a cyclic peptide inhibitor. Finally, this chapter presents the use of molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV main protease and the highly homologous SARS-CoV-2 main protease.The supporting information section of this chapter provides an illustrated step-by-step protocol showing the inhibitor design process. These and other molecular modeling studies helped our laboratory decide to pursue the synthesis of the cyclic peptide and experimentally evaluate its promise as an inhibitor of SARS-CoV-2 main protease. The molecular modeling studies presented in this chapter may help students and scientists design their own drug candidates for COVID-19 and the coronaviruses that may cause future pandemics.

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