SciCombinator

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Concept: Peptide bond

168

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

Concepts: DNA, Proteins, Protein, Molecular biology, Peptide bond, Protein biosynthesis, Molecular genetics, Intein

162

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

Concepts: Protein, Protein structure, Amino acid, Signal transduction, Peptide bond, Grb2, SH2 domain, Complex analysis

44

Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen-DR4(DRB1*0401)-restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group’s daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated β cell-specific CD8 T cells, and favorable β cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in β cell function, and is associated with antigen-specific and nonspecific immune modulation.

Concepts: Immune system, Protein, Insulin, Diabetes mellitus, Diabetes mellitus type 1, Immunology, Peptide bond, Cytotoxic T cell

37

The prebiotic replication of information-coding molecules is a central problem concerning life’s origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH2 yields the isotactic product FRFRFRFR-NH2, 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH2 and Ac-VDVDVDVDV-NH2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.

Concepts: Protein, Amino acid, Acid, Peptide bond, Stereochemistry, Peptide, Amyloid, Peptide synthesis

33

γ-secretase inhibitors (GSI) are drugs developed to decrease amyloid-β peptide (Aβ) production by inhibiting intramembranous cleavage of β-amyloid protein precursor (βAPP). However, a large phase 3 trial of semagacestat, a potential non-transition state analog (non-TSA) GSI, in patients with Alzheimer’s disease (AD) was terminated due to unexpected aggravation of cognitive deficits and side effects. Here, we show that some semagacestat effects are clearly different from a phenotype caused by a loss of function of presenilins, core proteins in the γ-secretase complex. Semagacestat increases intracellular byproduct peptides, produced along with Aβ through serial γ-cleavage of βAPP, as well as intracellular long Aβ species, in cell-based and in vivo studies of AD model mice. Other potential non-TSA GSIs, but not L685,458, a TSA GSI, have similar effects. Furthermore, semagacestat inhibits release of de novo intramembranous γ-byproducts to the soluble space. Thus, semagacestat is a pseudo-GSI, and therefore, the semagacestat clinical trial did not truly test the Aβ hypothesis.

Concepts: Alzheimer's disease, Protein, Protein structure, Clinical trial, Peptide bond, Peptide, Inhibitor, Amyloid precursor protein

32

Culture-based blood-brain barrier (BBB) models are crucial tools to enable rapid screening of brain-penetrating drugs. However, reproducibility of in vitro barrier properties and permeability remain as major challenges. Here, we report that self-assembling multicellular BBB spheroids display reproducible BBB features and functions. The spheroid core is comprised mainly of astrocytes, while brain endothelial cells and pericytes encase the surface, acting as a barrier that regulates transport of molecules. The spheroid surface exhibits high expression of tight junction proteins, VEGF-dependent permeability, efflux pump activity and receptor-mediated transcytosis of angiopep-2. In contrast, the transwell co-culture system displays comparatively low levels of BBB regulatory proteins, and is unable to discriminate between the transport of angiopep-2 and a control peptide. Finally, we have utilized the BBB spheroids to screen and identify BBB-penetrant cell-penetrating peptides (CPPs). This robust in vitro BBB model could serve as a valuable next-generation platform for expediting the development of CNS therapeutics.

Concepts: Protein, Brain, Angiogenesis, Peptide bond, Brain tumor, Peptide, Astrocyte, Blood-brain barrier

31

One of the most important reactions in organic chemistry–amide bond formation–is often overlooked as a contemporary challenge because of the widespread occurrence of amides in modern pharmaceuticals and biologically active compounds. But existing methods are reaching their inherent limits, and concerns about their waste and expense are becoming sharper. Novel chemical approaches to amide formation are therefore being developed. Here we review and summarize a new generation of amide-forming reactions that may contribute to solving these problems. We also consider their potential application to current synthetic challenges, including the development of catalytic amide formation, the synthesis of therapeutic peptides and the preparation of modified peptides and proteins.

Concepts: Protein, Oxygen, Amino acid, Chemical reaction, Peptide bond, Nitrogen, Amide, Peptide synthesis

28

We present a computational pipeline for the quantification of peptides and proteins in label-free LC-MS/MS datasets. The pipeline is composed of tools from the OpenMS software framework and is applicable to the processing of large experiments (50+ samples). We describe several enhancements that we have introduced to OpenMS to realize the implementation of this pipeline. They include new algorithms for centroiding of raw data, for feature detection, for the alignment of multiple related measurements, and a new tool for the calculation of peptide and protein abundances. Where possible, we compare the performance of the new algorithms to that of their established counterparts in OpenMS. We validate the pipeline based on two small datasets that provide ground truths for the quantification. There, we also compare our results to those of MaxQuant and Progenesis LC-MS – two popular alternatives for the analysis of label-free data. We then show how our software can be applied to a large heterogenous dataset of 58 LC-MS/MS runs.

Concepts: Protein, Protein structure, Bioinformatics, Amino acid, Mass spectrometry, Peptide bond, Peptide, Proteomics

28

Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes.

Concepts: Protein, Amino acid, Cell biology, Peptide bond, Bacillus, Bacillus subtilis, Proline, Osmoprotectant

27

A great deal of effort has been invested in the design and characterization of systems which spontaneously assemble into nanofibers. These systems are interesting for their fundamental supramolecular chemistry and have also been shown to be promising materials, particularly for biomedical applications. Multidomain peptides are one such assembler, and in previous work we have demonstrated the reversibility of their assembly under mild and easily controlled conditions, along with their utility for time-controlled drug delivery, protein delivery, cell encapsulation, and cell delivery applications. Additionally, their highly compliant criteria for sequence selection allows them to be modified to incorporate protease susceptibility and biological-recognition motifs for cell adhesion and angiogenesis. However, control of their assembly has been limited to the formation of disorganized nanofibers. In this work, we expand our ability to manipulate multidomain-peptide assembly into parallel-aligned fiber bundles. Albeit this alignment is achieved by the shearing forces of syringe delivery, it is also dependent on the amino acid sequence of the multidomain peptide. The incorporation of the amino acid DOPA (3,4-dihydroxyphenylalanine) allows the self-assembled nanofibers to form an anisotropic hydrogel string under modest shear stress. The hydrogel string shows remarkable birefringence, and highly aligned nanofibers are visible in scanning electronic microscopy. Furthermore, the covalent linkage induced by DOPA oxidation allows covalent capture of the aligned nanofiber bundles, enhancing their birefringence and structural integrity.

Concepts: Protein, Protein structure, Amino acid, Peptide bond, Peptide, Force, Shear stress, Assembly language