Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

Peptide stapling by late-stage Suzuki–Miyaura cross-coupling

The development of peptide stapling techniques to stabilise α-helical secondary structure motifs of peptides led to the design of modulators of protein–protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki–Miyaura cross-coupling of bromotryptophan-containing peptides of the catenin-binding domain of axin. Optimisation of the linker length in order to find a compromise between both sufficient linker rigidity and flexibility resulted in a peptide with an increased α-helicity and enhanced binding affinity to its native binding partner β-catenin. An increased proteolytic stability against proteinase K has been demonstrated.

Dioctanoyl Ultrashort Tetrabasic β-Peptides Sensitize Multidrug-Resistant Gram-Negative Bacteria to Novobiocin and Rifampicin

Recently reported peptidomimetics with increased resistance to trypsin were shown to sensitize priority multidrug-resistant (MDR) Gram-negative bacteria to novobiocin and rifampicin. To further optimize proteolytic stability, β-amino acid-containing derivatives of these compounds were prepared, resulting in three dioctanoyl ultrashort tetrabasic β-peptides (dUSTBβPs). The nonhemolytic dUSTBβP 3, comprised of three β3-homoarginine residues and two fatty acyl tails eight carbons long, enhanced the antibacterial activity of various antibiotics from different classes. Notably, compound 3 retained the ability to potentiate novobiocin and rifampicin in wild-type Gram-negative bacteria against MDR clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. dUSTBβP 3 reduced the minimum inhibitory concentration of novobiocin and rifampicin below their interpretative susceptibility breakpoints. Furthermore, compound 3 exhibited improved in vitro stability (86.8 ± 3.7% remaining) relative to its α-amino acid-based counterpart (39.5 ± 7.4% remaining) after a 2 h incubation in human plasma.

Identifying signatures of proteolytic stability and monomeric propensity in O-glycosylated insulin using molecular simulation

Insulin has been commonly adopted as a peptide drug to treat diabetes given its ability to facilitate the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin’s proteolytic stability and reduce its dimerization propensity, but the understanding of such phenomena at the molecular level is still evasive. To address this challenge, we propose and test several structural determinants that could potentially in uence insulin’s proteolytic stability and dimerization propensity. We used these as the metrics to assess the properties of interest from 10 s aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, as was in part the solvent accessible surface area of proteolytic sites, while other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation in uences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.

Antiseptic 9-Meric Peptide with Potency against Carbapenem-Resistant Acinetobacter baumannii Infection

Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.

Protein Engineering with A Glycosylation Circuit Enables Improved Enzyme Characteristics

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

Identifying signatures of proteolytic stability and monomeric propensity in O-glycosylated insulin using molecular simulation

Insulin has been commonly adopted as a peptide drug to treat diabetes given its ability to facilitate the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin’s proteolytic stability and reduce its dimerization propensity, but the understanding of such phenomena at the molecular level is still evasive. To address this challenge, we propose and test several structural determinants that could potentially in uence insulin’s proteolytic stability and dimerization propensity. We used these as the metrics to assess the properties of interest from 10 s aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, as was in part the solvent accessible surface area of proteolytic sites, while other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation in uences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.