Protease Susceptibility Research Articles

Global profiling of protein complex dynamics with an experimental library of protein interaction markers.

Methods to systematically monitor protein complex dynamics are needed. We introduce serial ultrafiltration combined with limited proteolysis-coupled mass spectrometry (FLiP-MS), a structural proteomics workflow that generates a library of peptide markers specific to changes in PPIs by probing differences in protease susceptibility between complex-bound and monomeric forms of proteins. The library includes markers mapping to protein-binding interfaces and markers reporting on structural changes that accompany PPI changes. Integrating the marker library with LiP-MS data allows for global profiling of protein-protein interactions (PPIs) from unfractionated lysates. We apply FLiP-MS to Saccharomyces cerevisiae and probe changes in protein complex dynamics after DNA replication stress, identifying links between Spt-Ada-Gcn5 acetyltransferase activity and the assembly state of several complexes. FLiP-MS enables protein complex dynamics to be probed on any perturbation, proteome-wide, at high throughput, with peptide-level structural resolution and informing on occupancy of binding interfaces, thus providing both global and molecular views of a system under study.

Probing the Effects of Chirality on Self-Assembling Peptides: Hydrogel Formation, Degradation, Antigen Release, and Adjuvancy.

Multidomain peptides (MDPs) are amino acid sequences that self-assemble to form supramolecular hydrogels under physiological conditions that have shown promise for a number of biomedical applications. K2(SL)6K2 ("K2"), a widely studied MDP, has demonstrated the ability to enhance the humoral immune response to co-delivered antigen. Herein, we sought to explore the in vitro and in vivo properties of a peptide with the same sequence but opposite chirality (D-K2) since peptides composed of D-amino acids are resistant to protease degradation and potentially more immunostimulatory than their canonical counterparts. K2 and D-K2 hydrogels were characterized and evaluatedin vitro usingcircular dichroism, rheology, cryo-electron microscopy, andfluorescence recovery after photobleaching studies. In vivo experiments in SKH-1 mice were conducted to evaluateboth ovalbumin release from the hydrogels andhydrogel degradation. The injection site of the hydrogels was analyzed using histologyandhumoral immunity was assessed byELISA. In vitro, the enantiomeric hydrogels exhibited similar rheological properties, and fluorescence recovery after photobleaching experiments demonstrated that the diffusion of ovalbumin (OVA), a model antigen, was similar within both hydrogels. In vivo, K2 and D-K2 peptide hydrogels had similar OVA release rates, both releasing 89% of the antigen within 8days. Both hydrogels elicited a similar antigen-specific humoral immune response. However, the in vivo degradation of the D-K2 hydrogel progressed significantly slower than K2. After 4weeks in vivo, only 23 ± 7% of the K2 hydrogel remained at the injection site compared to 94 ± 7% of the D-K2 hydrogel, likely due to their different protease susceptibilities. Taken together, these data suggest that peptide chirality can be a useful tool for increasing hydrogel residence time for biomedical applications that would benefit from long persistence times and that, if an antigen releases over a sufficiently short period, release can be largely independent of degradation rate, though slower-diffusing payloads may exhibit degradation rate dependence. The online version contains supplementary material available at 10.1007/s12195-024-00806-1.

Systematic identification of structure-specific protein–protein interactions

The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis–mass spectrometry (LiP–MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP–MS detects well-characterized PPIs, including antibody–target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson’s disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

Polylactic Glycolic Acid-Mediated Delivery of Plectasin Derivative NZ2114 in Staphylococcus epidermidis Biofilms.

Antimicrobial peptides (AMPs) are antibiotic candidates; however, their instability and protease susceptibility limit clinical applications. In this study, the polylactic acid-glycolic acid (PLGA)-polyvinyl alcohol (PVA) drug delivery system was screened by orthogonal design using the double emulsion-solvent evaporation method. NZ2114 nanoparticles (NZ2114-NPs) displayed favorable physicochemical properties with a particle size of 178.11 ± 5.23 nm, polydispersity index (PDI) of 0.108 ± 0.10, ζ potential of 4.78 ± 0.67 mV, actual drug-loading rate of 4.07 ± 0.37%, encapsulation rate of 81.46 ± 7.42% and cumulative release rate of 67.75% (120 h) in PBS. The results showed that PLGA encapsulation increased HaCaT cell viability by 20%, peptide retention in 50% serum by 24.12%, and trypsin tolerance by 4.24-fold. Meanwhile, in vitro antimicrobial assays showed that NZ2114-NPs had high inhibitory activity against Staphylococcus epidermidis (S. epidermidis) (4-8 μg/mL). Colony counting and confocal laser scanning microscopy (CLSM) confirmed that NZ2114-NPs were effective in reducing the biofilm thickness and bacterial population of S. epidermidis G4 with a 99% bactericidal rate of persister bacteria, which was significantly better than that of free NZ2114. In conclusion, the results demonstrated that PLGA nanoparticles can be used as a reliable NZ2114 delivery system for the treatment of biofilm infections caused by S. epidermidis.

Tuning of hydrophobic–hydrophilic balance for the development of a salt-tolerant and protease-resistant lipopeptide AMP

Hydrophobic-hydrophilic balance of lipopeptides was tuned to generate a non-cytotoxic, salt-tolerant, and protease-resistant AMP, P18.

Incorporating a β-hairpin sequence motif to increase intracellular stability of a peptide-based PROTAC

Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of “undruggable” targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work identified peptides containing a β-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. The goal of this study was to investigate if a β-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and the CPP needed for PROTAC uptake and function. The degradation of the protein Tau was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the β-hairpin sequence. A series of time- and concentration-dependent studies confirmed that the β-hairpin sequence was an adequate alternative CPP and degron to facilitate the proteasome-mediated degradation of Tau. Microscopy studies confirmed the time-dependent uptake of the PROTAC and a degradation assay confirmed that the β-hairpin conjugated PROTAC had a greater lifetime in cells.

Design, synthesis, and characterization of type I collagen mimetic peptides.

The current wound-healing collagen mimetic peptides (CMPs) have limitations such as poor membrane permeability and protease susceptibility. Herein, the solid-phase peptide synthesis of CMPs containing the integrin binding motif GFOGER is reported. The peptide sequences also consist of lipophilic moieties (adamantane and palmitic acid) for improved membrane permeability and different collagen-inducing tripeptides, namely, Thr-Thr-Lys (TTK), Gly-His-Lys (GHK), Gln-Pro-Arg (QPR), and Glu-Glu-Met (EEM). The synthesized peptides were successfully characterized and purified using liquid chromatography-mass spectrometry and preparative high-performance liquid chromatography techniques, respectively. The palmitic acid moiety increased the hydrophobic nature of the peptides, and they were retained longer on the stationary material of the reverse phase C-18 column. The three-dimensional parallel-strand helical structure of peptide DGD-GG-GFOGER-GG-TTK-palmitate was obtained using nuclear magnetic resonance spectroscopy and circular dichroism. The synthesized peptides have the desired helical structure, which can promote integrin binding.

Changes of in vitro digestion rate and antioxidant activity of digestion products of ethanol-modified whey protein isolates

Effects of ethanol treatment at different concentrations (20%, 40%, 60%, and 80%, v/v) on in vitro digestion rate and antioxidant activity of digestion products of whey protein isolates (WPI) were investigated. The results indicated that treatment with an intermediate concentration of ethanol (40%, v/v) produced the highest WPI digestion rate and the strongest antioxidant activities of its digestion products ( P < 0.05), which was further verified by the results of microstructure, particle size and amino acid profiles of the digestion products. However, larger aggregates induced by higher ethanol concentrations (60 or 80%, v/v) might decrease the susceptibility of protease and subsequently lower the digestibility of WPI. The results suggested that a moderate concentration of ethanol treatment could be used as a potential way to enhance the in vitro digestion rate of WPI, as well as increase the antioxidant activities of its digestion products. • Ethanol treatment led to unfolding and aggregation of whey protein isolates (WPI). • Moderate ethanol concentration (EC) treatment had highest digestion rate of WPI. • Moderate EC promote the antioxidant activities of digestion products of WPI. • Higher EC led to larger aggregates and decrease the digestibility of WPI.

Antimicrobial susceptibility and production of virulence factors by bacteria recovered from bitches with pyometra.

Pyometra is one of the most common diseases in adult female dogs, characterized by a suppurative bacterial infection of the uterus with accumulation of inflammatory exudate and a variety of local and systemic clinical manifestations. This study aimed to identify the bacteria within the uterine content and vaginal canal of bitches with pyometra and evaluate their antimicrobial susceptibility and production of virulence factors. Uterine and vaginal content were collected with sterile swabs from 30 bitches diagnosed with pyometra. Bacteria were identified and assessed for their antimicrobial susceptibility and production of virulence factors, including biofilms, siderophores, proteases and hemolysins, both in planktonic and biofilm forms. A total of 82 bacterial isolates (35 uterus, 47 vagina), belonging to 21 species, were identified, with Escherichia coli as the most prevalent species (32/82, 39%). As for susceptibility, 39/79 (49.4%) isolates were resistant to one or more drugs, with resistance proportion among Gram-positive bacteria (87.5%) higher (p < .05) than that observed for Gram-negative bacteria (32.7%). Four coagulase-negative Staphylococcus species were resistant to methicillin. Regarding virulence, the isolates had low production of biofilms, siderophores, proteases and hemolysins, suggesting that the occurrence of pyometra might be more associated with host-related factors than bacterial virulence.

Impact of Food Matrices on Digestibility of Allergens and Poorly Allergenic Homologs.

BackgroundProtease resistance is considered a risk factor for allergenicity of proteins, although the correlation is low. It is nonetheless a part of the weight-of-evidence approach, proposed by Codex, for assessing the allergenicity risk of novel food proteins. Susceptibility of proteins to pepsin is commonly tested with purified protein in solution.ObjectiveFood proteins are rarely consumed in purified form. Our aim was to evaluate the impact of experimental and endogenous food matrices on protease susceptibility of homologous protein pairs with different degrees of allergenicity.MethodsPorcine and shrimp tropomyosin (ST) were subjected to sequential exposure to amylase, pepsin, and pancreatin in their respective endogenous matrix (pork tenderloin/boiled shrimp) and in three different experimental matrices (dessert mousse [DM], soy milk [SM], and chocolate bar [CB]). Digestion was monitored by immunoblotting using tropomyosin-specific antibodies. Recombinant peach and strawberry lipid transfer protein were biotinylated, spiked into both peach and strawberry fruit pulp, and subjected to the same sequential digestion protocol. Digestion was monitored by immunoblotting using streptavidin for detection.ResultsChocolate bar, and to a lesser extent SM, had a clear protective effect against pepsin digestion of porcine tropomyosin (PT) and to a lesser extent of ST. Increased resistance was associated with increased protein content. Spiking experiments with bovine serum albumin (BSA) confirmed the protective effect of a protein-rich matrix. The two tropomyosins were both highly resistant to pepsin in their protein-rich and lean native food matrix. Pancreatin digestion remained rapid and complete, independent of the matrix. The fat-rich environment did not transfer protection against pepsin digestion. Spiking of recombinant peach and strawberry lipid transfer proteins into peach and strawberry pulp did not reveal any differential protective effect that could explain differences in allergenicity of both fruits.ConclusionsProtein-rich food matrices delay pepsin digestion by saturating the protease. This effect is most apparent for proteins that are highly pepsin susceptible in solution. The inclusion of food matrices does not help in understanding why some proteins are strong primary sensitizers while homologs are very poor allergens. Although for induction of symptoms in food allergic patients (elicitation), a protein-rich food matrix that may contribute to increased risk, our results indicate that the inclusion of food matrices in the weight-of-evidence approach for estimating the potential risks of novel proteins to become allergens (sensitization), is most likely of very limited value.

Disulfide-Mediated Elongation of Amyloid Fibrils of α-Synuclein For Use in Producing Self-Healing Hydrogel and Dye-Absorbing Aerogel

Due to their mechanical robustness, biocompatibility, and nanoscale size, amyloid fibrils (AFs) have been considered as a potential nanomaterial for biological applications. Unfortunately, however, AFs are usually not fully extended because of their pre-mature breakage, which hampers their use to generate biocompatible suprastructures, although the amounts of AFs could be amplified via their self-propagation property. Here, we have demonstrated the full extension of AFs of α-synuclein (αS) by introducing a cysteine residue to its C-terminus which prevents the shear-induced fragmentation of AFs via site-directed disulfide bond formation on the exposed surface of AFs. These heat- and cold-resistant elongated AFs were entangled into self-healable hydrogels through a mild disulfide-exchange process in the presence of tris(2-carboxyethyl) phosphine, which subsequently developed into dye-absorbing aerogels upon freeze-drying without collapsing the three-dimensional internal fibrillar network. The resulting αS aerogel with high porosity and increased surface area was shown to be capable of absorbing both hydrophilic and hydrophobic substances. In addition, the aerogel was further engineered with 8-arm polyethylene glycol containing a sulfhydryl group to increase its drug loading capacity and protease susceptibility for drug unloading. The elongated AFs, therefore, have been suggested to play a pivotal component for the development of bio-nano-matrix for diverse biological applications including drug delivery, tissue engineering, and environmental remediation. Statement of significanceDue to accurate protein self-assembly process, α-synuclein forms an amyloid fibril which are the major component of Lewy bodies. In general, α-synuclein amyloid fibrils break under thermal fluctuations as these nanofibrils elongate to reach certain length. In this study, we have demonstrated the full extension of α-synuclein amyloid fibrils by introducing a cysteine residue to its C-terminus by forming site-directed disulfide bonds on the exposed surface of amyloid fibrils for the first time. The resulting elongated amyloid fibrils were mechanically robust and stable. By using elongated amyloid fibrils, we have made self-healable amyloid fibril hydrogel and dye-absorbing aerogel.

Glycosylation of FGFR4 in cholangiocarcinoma regulates receptor processing and cancer signaling.

Recent advances in targeted treatment for cholangiocarcinoma have focused on fibroblast growth factor (FGF) signaling. There are four receptor tyrosine kinases that respond to FGFs, and posttranslational processing has been demonstrated for each FGF receptor. Here, we investigated the role of N-linked glycosylation on the processing and function of FGFR4. We altered glycosylation through enzymatic deglycosylation, small molecule inhibition of glycosyltransferases, or through site-directed mutagenesis of selected asparagine residues in FGFR4. Signaling was tested through caspase activation, migration, and subcellular localization of FGFR4. Our data demonstrate that FGFR4 has multiple glycoforms, with predominant bands relating to the full-length receptor that has a high mannose- or hybrid-type form and a complex-type glycan form. We further identified a set of faster migrating FGFR4 bands that correspond to the intracellular kinase domain, termed FGFR4 intracellular domain (R4-ICD). These glycoforms and R4-ICD were detected in human cholangiocarcinoma tumor samples, where R4-ICD was predominant. Removal of glycans in intact cells by enzymatic deglycosylation resulted in increased processing to R4-ICD. Inhibition of glycosylation using NGI-1, an oligosaccharyltransferase inhibitor, reduced both high mannose- or hybrid- and complex-type glycan forms of FGFR4, increased processing and sensitized to apoptosis. Mutation of Asn-112, Asn-258, Asn-290, or Asn-311 to glutamine modestly reduced apoptosis resistance, while mutation of Asn-322 or simultaneous mutation of the other four asparagine residues caused a loss of cytoprotection by FGFR4. None of the glycomutants altered the migration of cancer cells. Finally, mutation of Asn-112 caused a partial localization of FGFR4 to the Golgi. Overall, preventing glycosylation at individual residues reduced the cell survival function of FGFR4 and receptor glycosylation may regulate access to an extracellular protease or proteolytic susceptibility of FGFR4.

Predicting and characterising protein damage in the extracellular matrix

Chronic UVR exposure of human skin can result in photo-ageing which manifests both externally and internally (as remodelling of skin layers including the extracellular matrix-rich dermis). However, the intermittent nature of UVR exposure over a timescale of decades combined with the longevity of many structural dermal proteins makes the identification of dermal photo-ageing targets and mechanisms challenging. Over the past ten years work in our group has demonstrated that: (i) proteins which are rich in amino acid chromophores are susceptible to physiologically relevant doses of solar simulated radiation, (ii) this protein degradation is mediated primarily by the photodynamic production of reaction oxygen species and (iii) UVR-chromophore rich proteins are located in tissue regions where they may act as endogenous sunscreens. We have also shown that ECM proteases selectively degrade UVR-damaged assemblies in vitro and have developed new machine learning-based models to predict protease cleavage sites and hence relative protease susceptibilities in the human skin proteome. The recent development of peptide location fingerprinting applied to conventional mass spectrometry datasets has allowed the identification of novel candidate biomarkers of UVR-induced damage as a consequence of photo-ageing. For example, this approach is able to identify structure-associated modifications in collagen VI alpha chains, although collagen VI remodeling is not evident by conventional histological analysis. These methods and resources (skin proteome database, protein susceptibility prediction and peptide location fingerprinting analysis) are available for use at www.manchesterproteome.manchester.ac.uk. Key areas for future research include using peptide location fingerprinting to: (i) characterise the structural hallmarks of ageing and photo-ageing mechanisms across different phototypes and (ii) evaluate the efficacy of rejuvenating treatments against novel protein biomarkers.

Fc-GDF15 glyco-engineering and receptor binding affinity optimization for body weight regulation

GDF15 is a distant TGF-β family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.

Glycosylation of recombinant rabbit immunoglobulins influences protease susceptibility as shown by comprehensive mass spectrometric glycan analysis.

Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Nonrecombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for nonrecombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance.

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Comparison of Hemoglobin Köln Erythrocyte Membranes With Malondialdehyde-Reacted Normal Erythrocyte Membranes

AbstractSplenectomized patients with hemoglobin (Hb) Köln have rigid RBCs with membrane polypeptide aggregates that are not dissociable with disulfide-reducing agents. Malondialdehyde (MDA) action on normal RBCs produced rigid RBCs with similar nondissociable aggregates. To test the hypothesis that Hb Köln RBC aggregates contained unsaturated MDA-type bonds, we reduced normal control RBC membranes, Hb Köln RBC membranes, and MDA-reacted membranes with [3H]NaBH4. Hb Köln RBC membranes and MDA-reacted membranes both had significantly more 3H incorporation than control membranes. Furthermore, 3H incorporation in both Hb Köln and MDA-treated membranes was located in the membrane polypeptide aggregates, presumably saturating the crosslinking bonds. After reaction of RBCs with [14C]MDA, the MDA label was similarly concentrated in the membrane polypeptide aggregates. Normal RBC membranes incubated with MDA were analyzed with and without reduction by NaBH4 prior to amino acid determination by high-performance liquid chromatography (HPLC). Reduction with NaBH4 after MDA treatment decreased the lysyl residues by 33% and the serine by 7% and increased by 10% the methionyl residues, but did not affect 12 other amino acids. Similar changes could be detected in NaBH4-reduced Hb Köln aggregates in methionine and serine content. MDA may also alter protein configuration, as evidenced by an increase in the protease susceptibility of membrane proteins from MDA-treated and Hb Köln RBCs. We conclude that Hb Köln RBC membranes, like MDA-treated membranes, have similar high molecular weight aggregates conferring decreased membrane deformability, [3H]NaBH4-reducible unsaturated bonds, changes in amino acid composition upon reduction, and protease-sensitive configurational changes.

Novel unbiased DARTS approach to find protein targets for endophyte extracts in a selected number of cancer cell lines

هدفت الدراسة الحالية إلى دراسة جدوى طريقة DARTS لتحديد ودراسة التفاعلات بين ربيطات البروتين، وإثبات فعالية الطريقة لتحديد البروتينات المستهدفة الجزيئية لبعض مقتطفات الخلايا الحيوية النشطة بيولوجيا. الفكرة الأساسية لهذا البحث هي دراسة إمكانية استخدام تقنية DARTS للكشف عن الأهداف الخلوية للمستخلصات الطبيعية قبل القيام بالتحليل الكيميائي لتحديد نوع الحاضنة التي توجد فيها. ولتحقيق الهدف من الدراسة، تم اختيار بعض العينات الطبيعية من الفطريات للدراسة، ثلاثة منها قادرة على وقف نمو ثلاثة أنواع من خلايا سرطان الدم وسرطان الرئة وسرطان عنق الرحم، كما يتضح من الاختبارات التي أجريت في هذه الدراسة. وبينت لنتائج أن استخدام تقنية DARTS لتحديد الأهداف الخلوية لهذه المقتطفات تم بنجاح. كما تم التعرف بالفعل على عدد من البروتينات التي تتفاعل مع هذه العينات داخل الخلية. كان من الممكن لأول مرة باستخدام DARTS متبوعًا بتحليل MALDI-TOF وبصمات الأصابع peptidmass لتحديد عدد من الجزيئات المستهدفة الخلوية للمستخلصات الحيوية النشطة من العينات الفطرية. الخلاصة: تساعد هذه الدراسة في تقليل عدد المستخلصات التي تدخل في عملية تطوير الدواء لأن المستخلصات غير المناسبة يتم استبعادها من البداية دون الحاجة إلى تحليل كيميائي لتحديد التركيب، مما يساعد على تقليل التكلفة والوقت الفترة التي تتطلبها عملية تطوير الدواء في المستقبل. هذا يفتح مجالًا جديدًا لاستخدام هذه التقنية في عملية مسح المستخلصات الطبيعية الأولية الخلوية دون الحاجة إلى تحليلها كيميائيًا مما يقلل من الجهد والتكاليف. أخيرًا، يمكن تحديد عدد من البروتينات المثيرة للاهتمام التي قد تحفز الباحثين في دراسة مستقبلية على التركيب الكيميائي لهذه العينات لتحديد المكون النشط في كل منها تمامًا.

Reply to Kumari and Singh, "Antibiofilm Activity of Small Molecules Produced by Staphylococcus epidermidis against Staphylococcus aureus".

We appreciated Kumari and Singh´s comments on our paper (11). Although we have continued trying to determine the identity of the bioactive molecules present in Staphylococcus epidermidis cell-free conditioned media (CFCM), we are yet to be successful. Initially, phenol-soluble modulins (PSMs) were a potential candidate. However, the protease treatment used in our study, which did not abrogate CFCM activity, was somewhat similar to the treatment used by Bojet et al. (1), where supernatant activity due to PSMs was abolished by addition of a Streptomyces griseus protease mixture at 1 mg/ml at 37°C for 30 min followed by heat inactivation (80°C for 20 min). In our study, we treated CFCM with proteinase K (20 mg/ml) for 1 h at 37°C and also performed heat inactivation (100°C for 40 min) without any reduction of antibiofilm activity. In an independent study, Li et al. (2) also abolished supernatant activity due to PSMs by treating it with proteinase K (100 μg/ml) at 37°C for 1 h. Although these conditions are similar to the ones used in our study, it is important to note that the proteinase K concentration employed was 200-fold lower than the one we used and was still sufficient to eliminate PSM activity. Nevertheless, Kumari and Singh are correct when they state that the conformation of PSM aggregates can affect protease susceptibility, and we agree that it might be worth checking the effect of purified S. epidermidis PSMs on Staphylococcus aureus biofilms. Even if PSMs are involved, we believe that, altogether, our data suggest that the bioactivity detected is multifactorial and may involve multiple molecules present in S. epidermidis CFCM. Our transcriptome sequencing (RNA-seq) results showed a huge impact of CFCM on gene expression, repressing the expression of genes associated with both biofilm formation and quorum sensing. These two activities are thought to be inversely regulated, and inhibition of quorum sensing is associated with increased biofilm formation (3–5). Previous work has shown that PSMs from the same species prevent biofilm formation and promote biofilm disruption in both S. aureus and S. epidermidis (6–9). If PSMs prove to be one of the antibiofilm molecules in S. epidermidis CFCM, it will be interesting to determine their mechanism of action and activity during various interspecies interactions, as has been shown by Kumari and Singh regarding Rhizopus arrhizus (10). We thank the authors for all the suggestions for experiments, and we believe that testing CFCM of an agr mutant strain of S. epidermidis will be a valuable addition to future work.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule.

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.