Protease-Resistant Peptides for Targeting and Intracellular Delivery of Therapeutics

Peptides show high promise in the targeting and intracellular delivery of next-generation bio- and nano-therapeutics. However, the proteolytic susceptibility of peptides is one of the major limitations of their activity in biological environments. Numerous strategies have been devised to chemically enhance the resistance of peptides to proteolysis, ranging from N- and C-termini protection to cyclization, and including backbone modification, incorporation of amino acids with non-canonical side chains and conjugation. Since conjugation of nanocarriers or other cargoes to peptides for targeting and cell penetration may already provide some degree of shielding, the question arises about the relevance of using protease-resistant sequences for these applications. Aiming to answer this question, here we provide a critical review on protease-resistant targeting peptides and cell-penetrating peptides (CPPs). Two main approaches have been used on these classes of peptides: enantio/retro-enantio isomerization and cyclization. On one hand, enantio/retro-enantio isomerization has been shown to provide a clear enhancement in peptide efficiency with respect to parent L-amino acid peptides, especially when applied to peptides for drug delivery to the brain. On the other hand, cyclization also clearly increases peptide transport capacity, although contribution from enhanced protease resistance or affinity is often not dissected. Overall, we conclude that although conjugation often offers some degree of protection to proteolysis in targeting peptides and CPPs, modification of peptide sequences to further enhance protease resistance can greatly increase homing and transport efficiency.

Developmental series of gene expression clarifies maternal mRNA provisioning and maternal-to-zygotic transition in a reef-building coral

Background Maternal mRNA provisioning of oocytes regulates early embryogenesis. Maternal transcripts are degraded as zygotic genome activation (ZGA) intensifies, a phenomenon known as the maternal-to-zygotic transition (MZT). Here, we examine gene expression over nine developmental stages in the Pacific rice coral, Montipora capitata, from eggs and embryos at 1, 4, 9, 14, 22, and 36 h-post-fertilization (hpf), as well as swimming larvae (9d), and adult colonies. Results Weighted Gene Coexpression Network Analysis revealed four expression peaks, identifying the maternal complement, two waves of the MZT, and adult expression. Gene ontology enrichment revealed maternal mRNAs are dominated by cell division, methylation, biosynthesis, metabolism, and protein/RNA processing and transport functions. The first MZT wave occurs from ~4-14 hpf and is enriched in terms related to biosynthesis, methylation, cell division, and transcription. In contrast, functional enrichment in the second MZT wave, or ZGA, from 22 hpf-9dpf, includes ion/peptide transport and cell signaling. Finally, adult expression is enriched for functions related to signaling, metabolism, and ion/peptide transport. Our proposed MZT timing is further supported by expression of enzymes involved in zygotic transcriptional repression (Kaiso) and activation (Sox2), which peak at 14 hpf and 22 hpf, respectively. Further, DNA methylation writing (DNMT3a) and removing (TET1) enzymes peak and remain stable past ~4 hpf, suggesting that methylome programming occurs before 4 hpf. Conclusions Our high-resolution insight into the coral maternal mRNA and MZT provides essential baseline information to understand parental carryover effects and the sensitivity of developmental success under increasing environmental stress.

Assessment of Bioavailability after In Vitro Digestion and First Pass Metabolism of Bioactive Peptides from Collagen Hydrolysates

Collagen hydrolysates (CHs) are composed of bioactive peptides (BAPs), which possess health enhancing properties. There is a knowledge gap regarding the bioavailability of these BAPs that involves intestinal transport and hepatic first pass effects. A simulated gastrointestinal model was used to generate digesta from two CHs (CH-GL and CH-OPT), which were applied to a novel transwell co-culture of human intestinal epithelium cell line-6 (HIEC-6) and hepatic (HepG2) cells to simulate in vivo conditions of absorption and first pass metabolism. Peptide transport, hepatic first pass effects, and bioavailability were determined by measuring BAPs (Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, Gly-Pro-Hyp) using an innovative capillary electrophoresis method. All peptides were transported across the intestinal cell layer to varying degrees with both CHs; however, Gly-Pro-Hyp was transported only with CH-GL, but not CH-OPT. Notable hepatic production was observed for Ala-Hyp with both CH treatments, and for Pro-Hyp and Gly-Pro with CH-GL only. All peptides were bioavailable (>10%), except for Gly-Pro-Hyp after CH-OPT. Overall, a high degree of transport and hepatic first pass effects on CH-derived BAPs were observed. Further research is needed to explore the hepatic mechanisms related to the production of BAPs and the bifunctional effects of the bioavailable BAPs noted in this study.

Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

ABSTRACTThe proton electrochemical gradient drives substrate transport across the cell membrane via a diverse set of secondary active transporters. Proton coupled peptide transporters (POTs) are important for peptide transport in prokaryotes and eukaryotic cells, where they mediate the uptake of di- and tri-peptides in addition to drug and pro-drug molecules. Previously, we captured a POT transporter from Staphylococcus hominis, PepTSh, in a cytoplasm-facing, inward open state (Minhas et al., 2018). Biochemical experiments have further revealed several critical residues for proton coupled transport; however, the precise role played by these residues in coupling proton binding to conformational changes as well as the timescales for proton transfers have remained obscure. Here, we employed multiscale modeling, including classical molecular dynamics, reactive molecular dynamics, and enhanced free energy sampling to characterize proton coupling within this transporter. We show directly that proton binding to a glutamate on TM7 opens the extracellular gate. The inward proton flow is found to induce movement of the peptide towards the cytosol by varying the protonation state of a second conserved glutamate on TM10. We also show that proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible, revealing a mechanism for proton movement inside the transporter.

Serotonin Exposure Improves Stress Resistance, Aggregation, and Biofilm Formation in the Probiotic Enterococcus faecium NCIMB10415

The role of the microbiota–gut–brain axis in maintaining a healthy status is well recognized. In this bidirectional flux, the influence of host hormones on gut bacteria is crucial. However, data on commensal/probiotics are scarce since most reports analyzed the effects of human bioactive compounds on opportunistic strains, highlighting the risk of increased pathogenicity under stimulation. The present investigation examined the modifications induced by 5HT, a tryptophan-derived molecule abundant in the intestine, on the probiotic Enterococcus faecium NCIMB10415. Specific phenotypic modifications concerning the probiotic potential and possible effects of treated bacteria on dendritic cells were explored together with the comparative soluble proteome evaluation. Increased resistance to bile salts and ampicillin in 5HT-stimulated conditions relate with overexpression of specific proteins (among which Zn-beta-lactamases, a Zn-transport protein and a protein involved in fatty acid incorporation into the membrane). Better auto-aggregating properties and biofilm-forming aptitude are consistent with enhanced QS peptide transport. Concerning interaction with the host, E. faecium NCIMB10415 enhanced dendritic cell maturation, but no significant differences were observed between 5HT-treated and untreated bacteria; meanwhile, after 5HT exposure, some moonlight proteins possibly involved in tissue adhesion were found in higher abundance. Finally, the finding in stimulated conditions of a higher abundance of VicR, a protein involved in two-component signal transduction system (VicK/R), suggests the existence of a possible surface receptor (VicK) for 5HT sensing in the strain studied. These overall data indicate that E. faecium NCIMB10415 modifies its physiology in response to 5HT by improving bacterial interactions and resistance to stressors.

Mechanistic insights into the inhibitory effect of theaflavins on virulence factors production in Streptococcus mutans

Streptococcus mutans is the primary etiological agent associated with cariogenic process. The present study aimed to investigate the antibacterial and anti-virulence activities of theaflavins (TFs) to Streptococcus mutans UA159 as well as the underlying mechanisms. The results showed that TFs were capable of suppressing the acid production, cell adherence, water-insoluble exopolysaccharides production, and biofilm formation by S. mutans UA159 with a dosage-dependent manner while without influencing the cell growth. By a genome-wide transcriptome analysis (RNA-seq), we found that TFs attenuated the biofilm formation of S. mutans UA159 by inhibiting glucosyltransferases activity and the production of glucan-binding proteins (GbpB and GbpC) instead of directly blocking the expression of genes coding for glucosyltransferases. Further, TFs inhibited the expression of genes implicated in peptidoglycan synthesis, glycolysis, lipid synthesis, two-component system, signaling peptide transport (comA), oxidative stress response, and DNA replication and repair, suggesting that TFs suppressed the virulence factors of S. mutans UA159 by affecting the signal transduction and cell envelope stability, and weakening the ability of cells on oxidative stress resistance. In addition, an upregulated expression of the genes involved in protein biosynthesis, amino acid metabolism, and transport system upon TFs treatment indicated that cells increase the protein synthesis and nutrients uptake as one self-protective mechanism to cope with stress caused by TFs. The results of this study increase our current understanding of the anti-virulence activity of TFs on S. mutans and provide clues for the use of TFs in the prevention of dental caries.

Transport of Dietary Anti-Inflammatory Peptide, γ-Glutamyl Valine (γ-EV), across the Intestinal Caco-2 Monolayer

The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (Papp) of 1.56 × 10−6 ± 0.7 × 10−6 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly (p < 0.01) enhanced the transport rate to 2.5 × 10−6 ± 0.6 × 10−6 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the Papp of γ-EV to 4.36 × 10−6 ± 0.16 × 10−6 cm/s (p < 0.001), while theaflavin 3′-gallate and Gly-Sar significantly decreased the Papp (p < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.

Developmental series of gene expression clarifies maternal mRNA provisioning and maternal-to-zygotic transition in the reef-building coral Montipora capitata

BackgroundMaternal mRNA provisioning of oocytes regulates early embryogenesis. Maternal transcripts are degraded as zygotic genome activation (ZGA) intensifies, a phenomenon known as the maternal-to-zygotic transition (MZT). Here, we examine gene expression over nine developmental stages in the Pacific rice coral, Montipora capitata, from eggs and embryos at 1, 4, 9, 14, 22, and 36 hours-post-fertilization (hpf), as well as swimming larvae (9d), and adult colonies.ResultsWeighted Gene Coexpression Network Analysis revealed four expression peaks, identifying the maternal complement, two waves of the MZT, and adult expression. Gene ontology enrichment revealed maternal mRNAs are dominated by cell division, methylation, biosynthesis, metabolism, and protein/RNA processing and transport functions. The first MZT wave occurs from ∼4-14 hpf and is enriched in terms related to biosynthesis, methylation, cell division, and transcription. In contrast, functional enrichment in the second MZT wave, or ZGA, from 22 hpf-9dpf, includes ion/peptide transport and cell signaling. Finally, adult expression is enriched for functions related to signaling, metabolism, and ion/peptide transport. Our proposed MZT timing is further supported by expression of enzymes involved in zygotic transcriptional repression (Kaiso) and activation (Sox2), which peak at 14 hpf and 22 hpf, respectively. Further, DNA methylation writing (DNMT3a) and removing enzymes (TET1) peak and remain stable past ∼4 hpf, indicating that methylome programming occurs before 4 hpf.ConclusionsOur high-resolution insight into the coral maternal mRNA and MZT provides essential information regarding setting the stage for, and the sensitivity of, developmental success and parental carryover effects under increasing environmental stress.

Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria

Over the past several decades, we have observed a very rapid development in the biotechnological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be widely used in various biotechnological processes. This article characterizes the proteolytic activity, describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Potential applications of proteolytic enzymes in many industries are also presented, including the food industry.