Degradation Of Peptides Research Articles

Impact of porcine brush border membrane enzymes on INFOGEST in vitro digestion model: A step forward to mimic the small intestinal phase

Brush border membrane (BBM) enzymes greatly affect the bioaccessibility and bioavailability of food nutrients. Despite their physiological importance, a step simulating the final stage of intestinal digestion has not yet been included in the harmonized protocols for in vitro digestion, primarily due to the challenges of replicating the dynamics of intestinal degradation. Herein, we propose an advancement toward a more physiologically relevant method, complementing the harmonized static gastric-duodenal digestion INFOGEST model with the missing small intestinal phase. BBM hydrolase activity, incubation time, at pH 7.2 were established to reproduce the small intestinal conditions. Skim milk powder, as a model of protein food, was subjected to the in vitro static digestion. Immediately after the duodenal phase, digesta were supplemented with BBM vesicles purified from pig jejunum. To comply with the dynamic nature of intestinal digestion and balance the spontaneous inactivation of hydrolases, BBM supplements were added every two hours throughout 6 h incubation time. Peptide degradation was monitored at each stage of digestion by amino acid analysis, free α-amino group assay, HPLC, LC-MS/MS. Hydrolysis by BBM peptidases led to a significant increase of free amino acids, reflecting the known level of amino acid adsorption (> 90 %) in humans after eating milk proteins. LC-MS/MS analysis demonstrated that BBM hydrolases erode progressively the peptides released by gastro-duodenal processing up to stable sequence motifs. The approach described is particularly relevant when the endpoint is identifying the peptide sequences that cannot be further hydrolysed by digestive enzymes or to determine the amino acid bio-accessibility.

IsoAsp-Quest: Workflow development for isoAsp identification using database searches.

A recent study reported that isomerization of aspartyl residues (Asp) occurs in various tissues and proteins in vivo. For a comprehensive analysis of post-translational modifications, the MS-based proteomic approach is a straightforward method; however, the isomerization of Asp does not alter its molecular weight. Therefore, a unique method is required to analyze Asp isomers using mass spectrometry. Herein, we present a novel strategy, isoAsp-Quest, which is a database search-oriented isoAsp identification method. isoAsp is specifically converted to 18O-labeled Lα-Asp by the enzymatic reaction of protein L-isoaspartyl-O-methyltransferase (PIMT) in 18O water with a mass shift of 2 Da, which, in principle, enables us to distinguish Asp isomers. However, in practice, a labeled Lα-Asp signal overlaps with that of endogenous Lα-Asp, making detection challenging. Therefore, degradation of the endogenous Lα-Asp peptide by AspN and subsequent removal of AspN were performed prior to the PIMT reaction. This strategy was applied to bovine lens α-crystallin. Consequently, several Asp isomerization sites, consistent with human αA-crystallin, were identified in bovine αA-crystallin, indicating that this strategy is also effective for biological proteins. Therefore, isoAsp-Quest enables the analysis of Lβ-Asp in a straightforward and rapid workflow, which may be useful for the quality control of protein products and biomarker discovery.

A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.

Peptides are widely used in biomaterials due to their easy of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for non-degradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. However, removing these constituents can be expensive, time consuming, and increases sample variability. Here we show that directly injecting samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration, and that hundreds of LC-MS runs can be done on a single column without a significantly diminish the ability to quantify the degradation of peptide libraries. We also show that column failure is evident when hydrophilic peptides fail to be retained on the column, and this can be easily identified using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.

Kinetic and Structural Studies of the Plastidial Solanum tuberosum Phosphorylase.

Kinetics and structural studies of the plastidial Solanum tuberosum phosphorylase (stPho1) revealed that the most active form of the enzyme (stPho1ΔL78) is composed by two segments generated by proteolytic degradation of an approximately 65-residue-long peptide (L78) approximately in the middle of the stPho1 primary structure. stPho1ΔL78 is 1.5 times more active than the nonproteolyzed enzyme in solution and shows stronger specificity for glycogen, α-d-glucose, caffeine, and β-cyclodextrin than stPho1. The crystal structure of stPho1ΔL78 has been resolved at 2.2 Å resolution and revealed similarities and differences with the mammalian enzymes. The structural fold is conserved as is the active site, while other binding sites such as the inhibitor, the glycogen storage, the quercetin, and the allosteric are not. The binding of α-d-glucose, caffeine, and β-cyclodextrin to stPho1 has been studied by X-ray crystallography and revealed significant differences from those of the mammalian phosphorylases. As stPho1 is capable of catalyzing both starch synthesis and degradation, our studies suggest that the direction of stPho1 activity is regulated by the proteolytic degradation of the L78 peptide.

Changes in urinary output due to concomitant administration of sacubitril/valsartan and atrial natriuretic peptide in patients with heart failure: a multicenter retrospective cohort study

BackgroundSacubitril/valsartan is an angiotensin receptor neprilysin inhibitor (ARNI) that inhibits the degradation of endogenous natriuretic peptides. Therefore, ARNIs may increase the efficacy of human atrial natriuretic peptide (hANP), a drug for acute heart failure, by mediating its pharmacological mechanism. This study was aimed at evaluating the effects of ARNIs on the pharmacological effects of hANP by using surrogate marker, such as urinary output, in patients with heart failure.MethodsIn this multicenter retrospective cohort study, adult patients with heart failure who were taking angiotensin II receptor blockers (ARB) or ARNIs combined with hANP were enrolled. Information on basic characteristics, clinical laboratory data, medical history, and severity of cardiac insufficiency were collected from electronic medical records. The primary outcome was the change in adjusted fluid balance, calculated by IN-volume (mL/day) – OUT-volume (mL/day) / daily hANP dosage (μg).ResultsNinety-two and 62 patients in the ARB + hANP and ARNI + hANP groups, respectively, were eligible for analysis. The adjusted fluid balance in the ARNI + hANP group was significantly lower than that in the ARB + hANP group (p = 0.001). After propensity score matching, 27 patients from each group were included. Similarly, there was a significant reduction in adjusted fluid balance in the ARNI + hANP group after propensity score matching (p = 0.026).ConclusionsThese findings suggest that ARNIs may enhance the efficacy of hANP and the combination of the two may be effective in the treatment of heart failure.

Mitigating In-Column Artificial Modifications in High-Temperature LC-MS for Bottom-Up Proteomics and Quality Control of Protein Biopharmaceuticals.

Elevating the column temperature is an effective strategy for improving the chromatographic separation of peptides. However, high temperatures induce artificial modifications that compromise the quality of the peptide analysis. Here, we present a novel high-temperature LC-MS method that retains the benefits of a high column temperature while significantly reducing peptide modification and degradation during reversed-phase liquid chromatography. Our approach leverages a short inline trap column maintained at a near-ambient temperature installed upstream of a separation column. The retentivity and dimensions of the trap column were optimized to shorten the residence time of peptides in the heated separation column without compromising the separation performance. This easy-to-implement approach increased peak capacity by 1.4-fold within a 110 min peptide mapping of trastuzumab and provided 10% more peptide identifications in exploratory LC-MS proteomic analyses compared with analyses conducted at 30 °C while maintaining the extent of modifications close to the background level. In the peptide mapping of biopharmaceuticals, where in-column modifications can falsely elevate the levels of some critical quality attributes, the method reduced temperature-related artifacts by 66% for N-terminal pyroGlu and 63% for oxidized Met compared to direct injection at 60 °C, thus improving reliability in quality control of protein drugs. Our findings represent a promising advancement in LC-MS methodology, providing researchers and industry professionals with a valuable tool for improving the chromatographic separation of peptides while significantly reducing the unwanted modifications.

Functional Peptides from Yak Milk Casein: Biological Activities and Structural Characteristics.

The average content of casein in yak milk is 40.2 g/L. Casein can be degraded by enzymatic digestion or food processing to produce abundant degradation peptides. International researchers have studied the degradation peptides of yak milk casein by using multiple techniques and methods, such as in vitro activity tests, cellular experiments, proteomics, bioinformatics, etc., and found that the degradation peptides have a wide range of functional activities that are beneficial to the human body, such as angiotensin-converting enzyme (ACE) inhibitory, antioxidant, anti-inflammatory, antidiabetic, antimicrobial, anticancer, and immunomodulatory activities, etc., and it has been proved that the types and strengths of functional activities are closely related to the structural characteristics of the peptides. This paper describes the characteristics of yak milk proteins, the functional activities, and mechanism of action of degraded peptides. Based on the types of functional activities of yak milk casein degradation peptides, we classified and elucidated the effects of structural factors, such as peptide molecular weight, peptide length, amino acid sequence, physicochemical properties, electrical charge, hydrophobicity, spatial conformation, chain length, and the type of enzyme on these activities. It reveals the great potential of yak milk casein degradation peptides as functional active peptide resources and as auxiliary treatments for diseases. It also provides important insights for analyzing yak casein degradation peptide activity and exploring high-value utilization.

Nitrogen Availability and Utilisation of Oligopeptides by Yeast in Industrial Scotch Grain Whisky Fermentation

Scotch grain whisky is produced with a substantial proportion of unmalted grains, which can result in nitrogen deficiency for yeast in the fermentable grain mash. This study examined nitrogen source availability and utilisation by three commercial whisky strains (Saccharomyces cerevisiae) during Scotch grain whisky fermentation, focusing on oligopeptides. Peptide uptake kinetics in synthetic whisky mash with defined peptides showed that oligopeptides of up to nine amino acids were taken up by the strains, albeit with some variability between the strains. The study found that peptides with appropriate molecular weights could replace free amino acids without negatively affecting fermentation kinetics. Moreover, fermentation performance improved when additional nitrogen was provided via peptides rather than diammonium phosphate. Analysis of industrial grain mash indicated that despite low initial yeast assimilable nitrogen, residual proteolytic activity from malt increased nitrogen availability during fermentation. Approximately 30% of the nitrogen consumed by yeast during grain mash fermentation was derived from peptides. LC-HRMS peptide analysis revealed complex dynamics of peptide formation, degradation, and utilisation. This study highlights the importance of oligopeptides in ensuring optimal fermentation efficiency in Scotch grain mash and similar substrates.

Enhanced Gut Microbiome Capacity for Amino Acid Metabolism is associated with Peanut Oral Immunotherapy Failure.

Peanut Oral Immunotherapy (POIT) holds promise for remission of peanut allergy, though treatment is protracted and successful in only a subset of patients. Because the gut microbiome is linked to food allergy, we sought to identify fecal microbial predictors of POIT efficacy and to develop mechanistic insights into treatment response. Longitudinal functional analysis of the fecal microbiome of children (n=79) undergoing POIT in a first double-blind, placebo-controlled clinical trial, identified five microbial-derived bile acids enriched in fecal samples prior to POIT initiation that predicted treatment efficacy (AUC 0.71). Failure to induce disease remission was associated with a distinct fecal microbiome with enhanced capacity for bile acid deconjugation, amino acid metabolism, and increased peanut peptide degradation in vitro . Thus, microbiome mechanisms of POIT failure appear to include depletion of immunomodulatory secondary bile and amino acids and the antigenic peanut peptides necessary to promote peanut allergy desensitization and remission.

Differential potassium channel inhibitory activities of a novel thermostable degradation peptide BmKcug1a-P1 from scorpion medicinal material and its N-terminal truncated/restored peptides

Thermally stable full-length scorpion toxin peptides and partially degraded peptides with complete disulfide bond pairing are valuable natural peptide resources in traditional Chinese scorpion medicinal material. However, their pharmacological activities are largely unknown. This study discovered BmKcug1a-P1, a novel N-terminal degraded peptide, in this medicinal material. BmKcug1a-P1 inhibited hKv1.2 and hKv1.3 potassium channels with IC50 values of 2.12 ± 0.27 μM and 1.54 ± 0.28 μM, respectively. To investigate the influence of N-terminal amino acid loss on the potassium channel inhibiting activities, three analogs (i.e., full-length BmKcug1a, BmKcug1a-P1-D2 and BmKcug1a-P1-D4) of BmKcug1a-P1 were prepared, and their potassium channel inhibiting activities on hKv1.3 channel were verified by whole-cell patch clamp technique. Interestingly, the potassium channel inhibiting activity of full-length BmKcug1a on the hKv1.3 channel was significantly improved compared to its N-terminal degraded form (BmKcug1a-P1), while the activities of two truncated analogs (i.e., BmKcug1a-P1-D2 and BmKcug1a-P1-D4) were similar to that of BmKcug1a-P1. Extensive alanine-scanning experiments identified the bonding interface (including two key functional residues, Asn30 and Arg34) of BmKcug1a-P1. Structural and functional dissection further elucidated whether N-terminal residues of the peptide are located at the bonding interface is important in determining whether the N-terminus significantly influences the potassium channel inhibiting activity of the peptide. Altogether, this research identified a novel N-terminal degraded active peptide, BmKcug1a-P1, from traditional Chinese scorpion medicinal material and elucidated how the N-terminus of peptides influences their potassium channel inhibiting activity, contributing to the functional identification and molecular truncation optimization of full-length and degraded peptides from traditional Chinese scorpion medicinal material Buthus martensii Karsch.

Quantifying and Controlling the Proteolytic Degradation of Cell Adhesion Peptides.

Peptides are widely used within biomaterials to improve cell adhesion, incorporate bioactive ligands, and enable cell-mediated degradation of the matrix. While many of the peptides incorporated into biomaterials are intended to be present throughout the life of the material, their stability is not typically quantified during culture. In this work, we designed a series of peptide libraries containing four different N-terminal peptide functionalizations and three C-terminal functionalizations to better understand how simple modifications can be used to reduce the nonspecific degradation of peptides. We tested these libraries with three cell types commonly used in biomaterials research, including mesenchymal stem/stromal cells (hMSCs), endothelial cells, and macrophages, and quantified how these cell types nonspecifically degraded peptides as a function of terminal amino acid and chemistry. We found that peptides in solution which contained N-terminal amines were almost entirely degraded by 48 h, irrespective of the terminal amino acid, and that degradation occurred even at high peptide concentrations. Peptides with C-terminal carboxylic acids also had significant degradation when cultured with the cells. We found that simple modifications to the termini could significantly reduce or completely abolish nonspecific degradation when soluble peptides were added to cells cultured on tissue culture plastic or within hydrogel matrices, and that functionalizations which mimicked peptide conjugations to hydrogel matrices significantly slowed nonspecific degradation. We also found that there were minimal differences in peptide degradation across cell donors and that sequences mimicking different peptides commonly used to functionalize biomaterials all had significant nonspecific degradation. Finally, we saw that there was a positive trend between RGD stability and hMSC spreading within hydrogels, indicating that improving the stability of peptides within biomaterial matrices may improve the performance of engineered matrices.

Oral Insulin Delivery: A Review on Recent Advancements and Novel Strategies.

Due to the lifestyle of people in the community in recent years, the prevalence of diabetes mellitus has increased, so New drugs and related treatments are also being developed. One of the essential treatments for diabetes today is injectable insulin forms, which have their problems and limitations, such as invasive and less admission of patients and high cost of production. According to the mentioned issues, Theoretically, Oral insulin forms can solve many problems of injectable forms. Many efforts have been made to design and introduce Oral delivery systems of insulin, such as lipid-based, synthetic polymer-based, and polysaccharide-based nano/microparticle formulations. The present study reviewed these novel formulations and strategies in the past five years and checked their properties and results. According to peer-reviewed research, insulin-transporting particles may preserve insulin in the acidic and enzymatic medium and decrease peptide degradation; in fact, they could deliver appropriate insulin levels to the intestinal environment and then to blood. Some of the studied systems increase the permeability of insulin to the absorption membrane in cellular models. In most investigations, in vivo results revealed a lower ability of formulations to reduce BGL than subcutaneous form, despite promising results in in vitro and stability testing. Although taking insulin orally currently seems unfeasible, future systems may be able to overcome mentioned obstacles, making oral insulin delivery feasible and producing acceptable bioavailability and treatment effects in comparison to injection forms.

Bioactive peptides released by lactic acid bacteria fermented pistachio beverages

Fruit and vegetable are the raw materials for many popular fermented beverages with potential health benefits. In the present study, a novel pistachio beverage fermented with selected strains of lactic acid bacteria belonging to the species Leuconostoc pseudomesenteroides and Companilactobacillus paralimentarius was produced, and the extent of proteolysis was evaluated. Physico-chemical and microbiological analyses, together with peptidomics and proteomics, were carried out on the beverage after 24 h of fermentation, using both the non-inoculated and chemically acidified beverage as controls. Amino acid sequence of hundreds of peptides mainly released from 2S albumin, 11S and 7S globulin were characterized. The number and frequency of identified peptides were higher in beverage started with Leuconostoc pseudomesenteroides, followed by Companilactobacillus paralimentarius and acidic beverage. These results suggest that, in addition to endogenous proteases active at acidic pH, the proteolytic system of LAB directly participated to peptide degradation to some extent. According to the BIOPEP database, a group of 31 peptides were potentially bioactive, and primarily associated with antioxidative properties, ACE and DPP-IV inhibition. The beverage started with Leuconostoc pseudomesenteroides showed the highest amount and number of bioactive peptides. This study lays the foundations for the design of novel pistachio-fermented beverage with potential health properties.

Encapsulation of Tenebrio molitor Hydrolysate with DPP-IV Inhibitory Activity by Electrospraying and Spray-Drying.

This study investigates the encapsulation of Tenebrio molitor hydrolysate exhibiting DPP-IV inhibitory activity by spray-drying and electrospraying techniques. First, we optimized the feed formulation and processing conditions required to obtain nano-microcapsules by electrospraying when using Arabic gum as an encapsulating agent and pullulan and Tween 20 as additives. The optimum formulation was also dried by spray-drying, where the removal of the additives was also assayed. Morphology analysis reveals that electrosprayed capsules have a smaller size (1.2 ± 0.5 µm vs. 12.4 ± 8.7 µm) and greater uniformity compared to those obtained by spray-drying. Regarding the surface nitrogen content and DPP-IV inhibitory activity, our results show no significant difference between the electrosprayed capsules and spray-dried capsules containing additives (IC50 of ~1.5 mg protein/mL). Therefore, it was concluded that adding additives during spray-drying allows for a similar encapsulation efficiency and reduced degradation during processing, as achieved by electrospraying technique but providing higher productivity. On the other hand, spray-dried capsules without additives displayed a higher surface nitrogen content percentage, which was mainly due to the absence of Tween 20 in the feed formulation. Consequently, these capsules presented a higher IC50 value (IC50 of 1.99 ± 0.03 mg protein/mL) due to the potential degradation of surface-exposed peptides.

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with “S134-H170-D198” catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min−1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme’s activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E.Key points• A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139.• Orf2 was involved in peptide metabolism both in vitro and in vivo.• Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Designing and synthesis of injectable hydrogel based on carboxymethyl cellulose/carboxymethyl chitosan containing QK peptide for femoral head osteonecrosis healing

Femoral head necrosis is a debilitating disorder that typically caused by impaired blood supply to the hip joint. In this study, a novel injectable hydrogel based on Oxidized Carboxymethyl Cellulose (OCMC)-Carboxymethyl Chitosan (CMCS) polymers containing an angiogenesis stimulator peptide (QK) with a non-toxic crosslinking interaction (Schiff based reaction) was synthesized to enhance angiogenesis following femoral head necrosis in an animal model. The physicochemical features of fabricated injectable hydrogel were analyzed by FTIR, swelling and degradation rate, rheometry, and peptide release. Also, the safety and efficacy were evaluated following an in vitro hydrogel injection study and an avascular necrosis (AVN) animal model. According to the results, the hydrogel exhibited an appropriate swelling ratio and water uptake (>90 %, 24 h) as well as a suitable degradation rate over 21 days accompanied by a continuous peptide release. Also, data showed that hydrogels containing QK peptide boosted the proliferation, differentiation, angiogenesis, and osteogenic potential of both Bone Marrow mesenchymal Stem Cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs) (****p < 0.0001 and ***p < 0.001, respectively). Furthermore, molecular and histological evaluations significantly demonstrated the overexpression of Runx2, Osteocalcin, Collagen I, VEGF and CD34 genes (**p < 0.01 and ***p < 0.001, respectively), and also femoral head necrosis was effectively prohibited, and more blood vessels were detected in defect area by OCMC-CMCS hydrogel containing QK peptide (bone trabeculae >9000, ***p < 0.001). In conclusion, the findings demonstrate that OCMC-CMCS-QK injectable hydrogel could be considered as an impressive therapeutic construct for femoral head AVN healing.

Degradation of the α-Carboxyl Terminus 11 Peptide: In Vivo and Ex Vivo Impacts of Time, Temperature, Inhibitors, and Gender in Rat.

In previous research, a synthetic α-carboxyl terminus 1 (αCT1) peptide derived from connexin 43 (Cx43) and its variant (αCT11) showed beneficial effects in an ex vivo ischemia-reperfusion (I/R) heart injury model in mouse. In an in vivo mouse model of cryo-induced ventricular injury, αCT1 released from adhesive cardiac patches reduced Cx43 remodeling and arrhythmias, as well as maintained cardiac conduction. Whether intravenous injection of αCT1 or αCT11 produces similar outcomes has not been investigated. Given the possibility of peptide degradation in plasma, this study utilized in vivo I/R cardiac injury and ex vivo blood plasma models to examine factors that may limit the therapeutic potential of peptide therapeutics in vivo. Following tail vein administration of αCT11 (100 μM) in blood, no effect on I/R infarct size was observed in adult rat hearts on day 1 (D1) and day 28 (D28) after injury (p > 0.05). There was also no difference in the echocardiographic ejection fraction (EF%) between the control and the αCT11 groups (p > 0.05). Surprisingly, αCT11 in blood plasma collected from these rats was undetectable within ∼10 min after tail vein injection. To investigate factors that may modulate αCT11 degradation in blood, αCT11 was directly added to blood plasma isolated from normal rats without I/R and peptide levels were measured under different experimental conditions. Consistent with in vivo observations, significant αCT11 degradation occurred in plasma within 10 min at 22 and 37 °C and was nearly undetectable by 30 min. These responses were reduced by the addition of protease/phosphatase (PTase/PPTase) inhibitors to the isolated plasma. Interestingly, no significant differences in αCT11 degradation in plasma were noted between male and female rats. We conclude that fast degradation of αCT11 is likely the reason that no beneficial effects were observed in the in vivo I/R model and inhibition or shielding from PTase/PPTase activity may be a strategy that will assist with the viability of peptide therapeutics.

Crystal structure of a newly identified M61 family aminopeptidase with broad substrate specificity that is solely responsible for recycling acidic amino acids.

Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP-dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure-function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3-7 amino acids. Further, we report the high-resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin-bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild-type and m61xc-knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N-termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post-proteasomal protein degradation in prokaryotes.

Structural insights into the potential binding sites of Cathepsin D using molecular modelling techniques

Alzheimer’s disease (AD) is the most prevalent type of dementia caused by the accumulation of amyloid beta (Aβ) peptides. The extracellular deposition of Aβ peptides in human AD brain causes neuronal death. Therefore, it has been found that Aβ peptide degradation is a possible therapeutic target for AD. CathD has been known to breakdown amyloid beta peptides. However, the structural role of CathD is not yet clear. Hence, for the purpose of gaining a deeper comprehension of the structure of CathD, the present computational investigation was performed using virtual screening technique to predict CathD's active site residues and substrate binding mode. Ligand-based virtual screening was implemented on small molecules from ZINC database against crystal structure of CathD. Further, molecular docking was utilised to investigate the binding mechanism of CathD with substrates and virtually screened inhibitors. Localised compounds obtained through screening performed by PyRx and AutoDock 4.2 with CathD receptor and the compounds having highest binding affinities were picked as; ZINC00601317, ZINC04214975 and ZINCC12500925 as our top choices. The hydrophobic residues Viz. Gly35, Val31, Thr34, Gly128, Ile124 and Ala13 help stabilising the CathD-ligand complexes, which in turn emphasises substrate and inhibitor selectivity. Further, MM-GBSA approach has been used to calculate binding free energy between CathD and selected compounds. Therefore, it would be beneficial to understand the active site pocket of CathD with the assistance of these discoveries. Thus, the present study would be helpful to identify active site pocket of CathD, which could be beneficial to develop novel therapeutic strategies for the AD.

Effect of PLGA raw materials on in vitro and in vivo performance of drug-loaded microspheres.

Poly(lactide-co-glycolide) and poly(lactic-co-glycolic acids) (PLGAs) play a critical role in the development of commercial long-acting injectable microsphere formulations. However, very little information is available describing the impact of PLGA manufacturer and monomer distribution along the polymer chain (e.g., glycolic blockiness (Rc) and average lactic block length (LL)) on the degradation and release behavior of PLGA drug carriers in vitro and in vivo. Here, we compared the in vitro and in vivo performance of (a) four leuprolide-loaded microsphere formulations prepared from similar low-molecular-weight acid-capped PLGAs (10-14 kD, i.e., Expansorb® DLG 75-2A, Purasorb® PDLG 7502A, Resomer® RG 752H and Wako® 7515) and (b) two triamcinolone acetonide-loaded (Tr-A) microsphere formulations from similar medium-molecular-weight ester-capped PLGAs (i.e., Expansorb® DLG 75-4E and Resomer® RG 753S). Lupron Depot® and Zilretta® were used as reference commercial products. The six 75/25 PLGAs displayed block lengths that were either above or below values expected from a random copolymer. Drug release and polymer degradation were monitored simultaneously in vitro and in vivo using a cage implant system. The four leuprolide-loaded formulations showed similar release and degradation patterns with some notable differences between each other. Microspheres from the Expansorb® polymer displayed lower LL and higher Rc relative to the other 3 PLGA 75/25 microspheres, and likewise exhibited distinct peptide release and degradation behavior compared to the other 3 formulations. For each formulation, leuprolide release was erosion-controlled up to about 30% release after the initial burst followed by a faster than erosion release phase. In vitro release was similar as that in vivo over the first phase but notably different from the latter release phase, particularly for the most blocky Expansorb® formulation. The Purasorb® and Wako® formulations displayed highly similar performance in release, degradation, and erosion analysis. By contrast, the two ester-capped Expansorb® DLG 75-4E and Resomer® RG 753S used to prepare Tr-A microspheres shared essentially identical LL and higher Rc and behaved similarly although the Expansorb® degraded and released the steroid faster in vivo, suggestive of other factors responsible (e.g., residual monomer). The in vivo release performance for both drugs from the six microsphere formulations was similar to that of the commercial reference products. In summary, this work details information on comparing the similarities and differences in in vitro and in vivo performance of drug-loaded microspheres as a function of manufacturing and microstructural variables of different types of PLGA raw materials utilized and could, therefore, be meaningful in guiding the source control during development and manufacturing of PLGA microsphere-based drug products. Future work will expand the analysis to include a broader range of LL and higher Rc, and add additional important formulation metrics (e.g., thermal analysis, and residual monomer, moisture, and organic solvent levels).