The effect of fatty diacid acylation of human PYY3-36 on Y2 receptor potency and half-life in minipigs

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide. However, it is not trivial to prolong the half-life of peptides by lipidation and still maintain high potency and good formulation properties. Here we show that attaching a fatty acid to the obesity-drug relevant peptide PYY3-36 is not sufficient for long pharmacokinetics (PK), since the position in the backbone, but also type of fatty acid and linker strongly influences PK and potency. Furthermore, understanding the proteolytic stability of the backbone is key to obtain long half-lives by lipidation, since backbone cleavage still occurs while associated to albumin. Having identified a PYY analogue with a sufficient half-life, we show that in combination with a GLP-1 analogue, liraglutide, additional weight loss can be achieved in the obese minipig model.

A novel esterase DacApva from Comamonas sp. strain NyZ500 with deacetylation activity for acetylated polymer polyvinyl alcohol

As a water-soluble polymer, the widely used polyvinyl alcohol (PVA) is produced from hydrolysis of polyvinyl acetate. Microbial PVA carbon backbone cleavage via a two-step reaction of dehydrogenation and hydrolysis has been well studied. Content of acetyl group is a pivotal factor affecting performance of PVA derivatives in industrial application, and deacetylation is a non-negligible part in PVA degradation. However, the genetic and biochemical studies of its deacetylation remain largely elusive. Here, Comamonas sp. strain NyZ500 was isolated for its capability of growing on acetylated PVA from activated sludge. A spontaneous PVA-utilization deficient mutant strain NyZ501 was obtained when strain NyZ500 was cultured in rich media. Comparative analysis between the genomes of these two strains revealed a fragment (containing a putative hydrolase gene dacApva) deletion in NyZ501 and dacApva-complemented strain NyZ501 restored the ability to grow on PVA. DacApva, which shares 21% identity with xylan esterase AxeA1 from Prevotella ruminicola 23, is a unique deacetylase catalyzing the conversion of acetylated PVA and its derivatives to deacetylated counterparts. This indicates that strain NyZ500 utilizes acetylated PVA via acetate as a carbon source to grow. DacApva also possessed the deacetylation ability for acetylated xylan and the antibiotic intermediate 7-aminocephalosporanic acid (7ACA) but the enzymes for the above two compounds had no activities against PVA derivatives. This study enhanced our understanding of the diversity of microbial degradation of PVA and DacApva characterized here is also a potential biocatalyst for the eco-friendly biotransformation of PVA derivatives and other acetylated compounds. IMPORTANCE: Water-soluble PVA, which possesses a very robust ability to accumulate in the environment, has a very grave environmental impact due to its widespread use in industrial and household applications. On the other hand, chemical transformation of PVA derivatives is currently being carried out at high energy consumption and high pollution conditions using hazardous chemicals (such as NaOH, methanol) under high temperatures. The DacApva reported here performs PVA deacetylation under mild conditions, then it has a great potential to be developed into an eco-friendly biocatalyst for biotransformation of PVA derivatives. DacApva also has deacetylation activity for compounds other than PVA derivatives, which facilitates its development into a broad-spectrum deacetylation biocatalyst for production of certain desired compounds.

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4–5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

Characterization of Protein Structure with Ion Mobility Mass Spectrometry, Multiplexed Fragmentation Strategies and Data Directed Analysis

<p>Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without <i>a priori</i> assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.</p>

Characterization of Protein Structure with Ion Mobility Mass Spectrometry, Multiplexed Fragmentation Strategies and Data Directed Analysis

<p>Activated ion mobility measurements provide Insights to the stability of tertiary and quaternary structures of proteins and pairing such approaches with fragmentation can delineate which part(s) of the primary sequence are disrupted from a folded structure. In this work we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to determine the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation as well as on collisionally activated photoproducts post ion mobility separation. For cytochrome c, there is a significant increase in the fragmentation yield with collisional activation post mobility, for all conformational states. Similar strategies are deployed with the model multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID leads to classic asymmetric charge distribution in subunit products, which when preceded by UV irradiation yields fragments from within the sub-unit that can be mapped to the quaternary fold. Data driven, multivariate analysis (MVA) was used to determine the significant differences in UVPD and CID fragmentation pattern following in source activation. This data driven approach reveals diagnostic fragments without <i>a priori</i> assignments limited to predicated backbone cleavage and provides a new approach to map conformation landscapes that may have wider utility.</p>

AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop

Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.

Effects of Cold Jet Atmospheric Pressure Plasma on the Structural Characteristics and Immunoreactivity of Celiac-Toxic Peptides and Wheat Storage Proteins

The present research reported the effects of structural properties and immunoreactivity of celiac-toxic peptides and wheat storage proteins modified by cold jet atmospheric pressure (CJAP) plasma. It could generate numerous high-energy excited atoms, photons, electrons, and reactive oxygen and nitrogen species, including O3, H2O2, •OH, NO2− and NO3− etc., to modify two model peptides and wheat storage proteins. The Orbitrap HR-LC-MS/MS was utilized to identify and quantify CJAP plasma-modified model peptide products. Backbone cleavage of QQPFP and PQPQLPY at specific proline and glutamine residues, accompanied by hydroxylation at the aromatic ring of phenylalanine and tyrosine residues, contributed to the reduction and modification of celiac-toxic peptides. Apart from fragmentation, oxidation, and agglomeration states were evaluated, including carbonyl formation and the decline of γ-gliadin. The immunoreactivity of gliadin extract declined over time, demonstrating a significant decrease by 51.95% after 60 min of CJAP plasma treatment in vitro. The CJAP plasma could initiate depolymerization of gluten polymer, thereby reducing the amounts of large-sized polymers. In conclusion, CJAP plasma could be employed as a potential technique in the modification and reduction of celiac-toxic peptides and wheat storage proteins.

Adaptation of Syntenic Xyloglucan Utilization Loci of Human Gut Bacteroidetes to Polysaccharide Side Chain Diversity

ABSTRACT Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomonas species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-l-arabinofuranosidases and GH95 alpha-l-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select Bacteroides. IMPORTANCE The catabolism of complex carbohydrates that otherwise escape the endogenous digestive enzymes of humans and other animals drives the composition and function of the gut microbiota. Thus, detailed molecular characterization of dietary glycan utilization systems is essential both to understand the ecology of these complex communities and to manipulate their compositions, e.g., to benefit human health. Our research reveals new insight into how ubiquitous members of the human gut microbiota have evolved a set of microheterogeneous gene clusters to efficiently respond to the structural variations of plant xyloglucans. The data here will enable refined functional prediction of xyloglucan utilization among diverse environmental taxa in animal guts and beyond.