Specific Peptide Research Articles

Untargeted fast proteomics analysis using UPLC-Orbitrap HRMS for halal authentication of meat and meat products.

The authenticity of halal meat is a global issue because pork adulteration occurs. Certain religions, such as Islam and Judaism, prohibit the use of pork in food products. The purpose of this study was to evaluate the volume of trypsin with 10, 50 and 100 μL (20 μg/100 μL) and the digestion time from overnight to 30-120 min to establish a fast and straightforward procedure on proteomic analysis for halal authentication of meat and meat products. The method was applied to raw meat and processed pork products. The results show that 30 min digestion time and 50 μL of trypsin could detect specific peptides from pork. The proteins such as L-lactate dehydrogenase (D2SW96), haemoglobin subunit beta (F1RII7), carbonic anhydrase (A0A4XIUCS1), and myosin-1 (A0A481AX92) could be the alternative protein that contains specific peptide from pork that could be used as peptide biomarker in raw pork meat. Two peptides from haemoglobin subunit beta (F1RII7) protein heat stable peptide biomarkers were not previously reported. The method is proven for fast analysis, simple protein extraction, and rapid identification of specific pork peptides in raw meat and processed pork products. SIGNIFICANCE: The authenticity of halal meat is a global issue because pork adulteration occurs. Certain religions, such as Islam and Judaism, prohibit the use of pork in food products. Pork is frequently used as an adulterant in high-quality, high-priced meats such as beef, fish meat slices, lamb, or other meat to increase profits because pork is less expensive than the other meats. The purpose of this study was to evaluate the volume of trypsin and the digestion time to establish a fast and straightforward procedure on proteomic analysis for halal authentication of meat and meat products. The results show that 30 min digestion time and 50 μL of trypsin could detect specific peptides from pork. The proteins such as L-lactate dehydrogenase (D2SW96), haemoglobin subunit beta (F1RII7), carbonic anhydrase (A0A4XIUCS1), and myosin-1 (A0A481AX92) could be the alternative protein that contains specific peptide from pork that could be used as peptide biomarker in raw pork meat. To the best of our knowledge, this is the first report that studied on fast analysis, simple protein extraction, and rapid identification of specific pork peptides in raw meat and processed pork products.

A comprehensive quantitative LC-MS/MS method for rapid gelatin source identification in food products: Comparison with PCR.

Authentication of gelatin sources are required for cultural beliefs and food integrity. This paper describes a sensitive and rapid detection of gelatin sources using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The specific peptide markers were adopted to accurately identify bovine and porcine gelatin in pharmaceutical capsules and jellies. Multiple reaction monitoring (MRM) was employed to identify and quantify over five specific peptide markers, each characterized by its unique precursor and product ion transitions. The developed method was validated at three concentration levels in gelatin-containing products to assess its accuracy and precision. The recovery (accuracy) of the proposed method was between 80% and 107%, and relative standard deviation (precision) was in the range of 5.16-9.97%. Linearity was obtained≥0.99 (R2). To ensure the accuracy of ingredient labeling, the LC-MS/MS results were compared with those obtained from PCR assays. The LC-MS/MS method demonstrated exceptional sensitivity, reliably detecting gelatin adulteration at concentrations as low as 0.01%. The developed LC-MS/MS method provides a rapid and accurate results for authenticating gelatin sources in various food products within 4hours.

P-1863. Maternal Mycobacterial-Specific T-cell Signatures and Infant Risk of Mycobacterium tuberculosis Infection

Abstract Background Tuberculosis disease (TB) caused 214,000 pediatric deaths in 2022. A growing body of evidence suggests that HIV exposed uninfected infants (iHEU) are at increased risk for Mycobacterium tuberculosis (Mtb) infection. Harnessing the power of the maternal immune system to protect infants has shown promise in other infections. Yet no well powered study has evaluated the association between maternal mycobacterial-specific T cell memory and infant protection from Mtb infection. To address this knowledge gap, we examined the hypothesis that previously undescribed maternal factors modulate infant immunity to bacillus Calmette-Guèrin (BCG) and Mtb.Fig 1.Mothers of infants negative for Mtb infection had higher proportions of CD8 T cells expressing IL2 in response to mycobacterial antigens.ESAT-6/CFP-10 negative median 0.004710 (95% CI 0.00096000-0.01103), positive median 0.0 (95% CI 0-0.009410). TBWCL negative median 0.01172 (95% CI 0.005340-0.01860), positive median 0.003710 (95% CI 0-0.008467). Methods This study was nested within a randomized controlled trial of isoniazid to prevent Mtb infection in 300 iHEU (iTIPS) who were vaccinated with BCG at birth, randomized to isoniazid or placebo at 6-10 weeks, and had Quantiferon-Plus (with IFNγ, IL2, IP10, TNF measured in supernatant) (QFT-4) at 14 months, and/or tuberculin skin testing (TST) at 14 and 24 months. Paired maternal peripheral blood mononuclear cells were collected at the 6-10 week visit. The majority (73%) of mothers started antiretroviral therapy before pregnancy, 26.3% during pregnancy, and 0.7% after pregnancy. Frequency of self-reported history of TB disease was 10.7%. We assessed the response of maternal T cells to Mtb whole cell lysate (TBWCL), a proxy for BCG, and ESAT-6/CFP-10 (E6C10), an Mtb specific peptide pool, by flow cytometry with a T cell panel including surface, memory, and activation markers and IL2, IL17A, IFNγ, and TNF. Results Our preliminary analysis of 166 of 235 maternal samples with flow cytometry revealed that mothers of infants with positive TST or QFT-4 had significantly fewer CD8+IL2+ cells in response to TBWCL (median 0.0037% vs 0.0117%, p=0.032) (Fig 1). Conversely mothers of these infants had significantly higher CD4+TNFa+ responses to E6C10 (median 0.0458% vs 0%, p=0.0014) and CD4+IFNγ+ responses to TBWCL (median 0.0533% vs 0.0302%, p=0.0395). Conclusion These results establish that maternal mycobacterial-specific immune signatures are associated with infant outcomes, which we plan to further investigate by comparing these maternal signatures with infant T cell response to TBWCL and determining the role of maternal microchimeric cells. Disclosures Sylvia M. LaCourse, MD, MPH, Merck: Grant/Research Support|UpToDate: Royalties

Dual surrogate imprinting: an innovative strategy for the preparation of virus-selective particles.

This work involves the preparation of dual surrogate-imprinted polymers (D-MIPs) for the capture of SARS-CoV-2. To achieve this goal, an innovative and novel dual imprinting approach using carboxylated-polystyrene (PS-COOH) nanoparticles with a diameter of 100 nm and a SARS-CoV-2 Spike-derived peptide was carried out at the surface of amine-functionalized silica (PS-NH2) microspheres with a diameter of 500 nm. Firstly, PS-COOH nanoparticles with the same size and spherical shape as the SARS-CoV-2 virus were employed to form hemispherical indentations (HI) at the surface of the PS-NH2 microspheres (obtaining dummy particle-imprinted polymers, HI-MIPs). Next, a specific peptide sequence representing the Spike protein at the surface of the target virus was also used as the second template to generate specific peptide binding sites at the HI. Finally, the PS-COOH and the peptide were removed by several washing steps providing D-MIPs, comprising both dummy particle indentations (HI) and peptide binding sites. The D-MIPs and HI-MIPs were in-depth characterized via scanning electron microscopy (SEM), transmission electron microscope (TEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), and energy dispersive X-ray analysis (EDAX). 100% rebinding efficiency was achieved for the SARS-CoV-2 peptide with D-MIPs highlighting its specificity vs. non-peptide-imprinted control polymers (HI-MIPs), which only achieved a binding efficiency of <40.5%. D-MIPs also showed higher affinity than HI-MIPs towards real SARS-CoV-2 virus. Furthermore, lower rebinding percentages for both HI-MIPs (8.5%) and D-MIPs (6.9%) were obtained when incubated with an alternative peptide (i.e., characteristic for Zika virus) indicating a successful peptide imprinting process for the target SARS-CoV-2 peptide.

QM Investigation of Rare Earth Ion Interactions with First Hydration Shell Waters and Protein-Based Coordination Models.

Conventional methods for extracting rare earth metals (REMs) from mined mineral ores are inefficient, expensive, and environmentally damaging. Recent discovery of lanmodulin (LanM), a protein that coordinates REMs with high-affinity and selectivity over competing ions, provides inspiration for new REM refinement methods. Here, we used quantum mechanical (QM) methods to investigate trivalent lanthanide cation (Ln3+) interactions with coordination systems representing bulk solvent water and protein binding sites. Energy decomposition analysis (EDA) showed differences in the energetic components of Ln3+ interaction with representatives of solvent (water, H2O) and protein binding sites (acetate, CH3COO-), highlighting the importance of accurate description of electrostatics and polarization in computational modeling of REM interactions with biological and bioinspired molecules. Relative binding free energies were obtained for Ln3+ with coordination complexes originating from binding sites in PDB structures of a lanthanum binding peptide (PDB entry 7CCO) and LanM, with explicit consideration of the first hydration shell waters, according to quasi-chemical theory (QCT). Beyond the first shell, the bulk solvent environment was represented with an implicit continuum model. Ln3+ interactions with (H2O)9 and both binding site models became more favorable, moving down the periodic series. This trend was more pronounced with the protein binding site models than with water, resulting in affinity increasing with periodic number, except for the last REM, Lu3+, which bound less favorably than the preceding element, Yb3+. Using the truncated 7CCO binding site model, the magnitude and trend of the experimental Ln3+ relative binding free energies for the whole 7CCO peptide were reproduced. Conversely, the previously reported experimental data for LanM show a preference for the earlier lanthanides; this is likely due to longer-range interactions and cooperative effects, which are not represented by the reduced models. Using the truncated 7CCO binding site model, the magnitude and trend of the experimental Ln3+ relative binding free energies for the whole 7CCO peptide were reproduced. In contrast to the previously reported experimental data for LanM, the peptide preferentially binds the earlier lanthanides. This difference likely arises due to longer-range interactions and cooperative effects not represented by the peptide. Further investigation of Ln3+ interactions with whole proteins using polarizable molecular mechanics models with explicit solvent is warranted to understand the influence of longer-ranged interactions, cooperativity, and bulk solvent. Nevertheless, the present work provides new insights into Ln3+ interactions with biomolecules and presents an effective computational platform for designing specific single-site REM binding peptides more efficiently.

Double Peptide-Functionalized Carboxymethyl Chitosan-Coated Liposomes Loaded with Dexamethasone as a Potential Strategy for Active Targeting Drug Delivery

Liposomes are intensively used as nanocarriers for biology, biochemistry, medicine, and in the cosmetics industry and their non-toxic and biocompatible nature makes these vesicles attractive systems for biomedical applications. Moreover, the conjugation of specific ligands to liposomes increases their cellular uptake and therapeutic efficiency. Considering these aspects, the aim of the present study was to obtain new formulations of cationic liposomes coated with dual-peptide functionalized carboxymethyl chitosan (CMCS) for the treatment of inner ear diseases. In order to achieve efficient active targeting and ensuring a high efficacy of the treatment, CMCS was functionalized with Tet1 peptide, to target specific ear cells, and TAT peptide, to ensure cellular penetration. Furthermore, dexamethasone phosphate was loaded as a model drug for the treatment of ear inflammation. The infrared spectroscopy confirmed the functionalization of CMCS with the two specific peptides. The mean diameter of the uncovered liposomes varied between 167 and 198 nm whereas the CMCS-coated liposomes ranged from 179 to 202 nm. TEM analysis showed the spherical shape and unilamellar structure of liposomes. The release efficiency of dexamethasone phosphate after 24 h from the uncoated liposomes was between 37 and 40% and it appeared that the coated liposomes modulated this release. The obtained results demonstrated that the liposomes are hemocompatible since, for a tested concentration of 100 µg/mL, the liposome suspension had a lysis of erythrocytes lower than 2.5% after 180 min of incubation. In addition, the peptide-functionalized CMCS-coated liposomes induced a non-significant effect on the viability of normal V79-4 cells after 48 h, at the highest doses. Values of 71.31% were recorded (CLCP-1), 77.28% (CLCP-2) and 74.36% (CLCP-3), correlated with cytotoxic effects of 28.69%, 22.72%, and 25.64%.

Target-specific peptides for BK virus agnoprotein identified through phage display screening: advancing antiviral therapeutics

BK virus is implicated in polyomavirus-associated nephropathy (PVAN) and hemorrhagic cystitis, particularly in kidney transplant recipients, affecting the functionality of the transplanted kidney and posing a risk of graft loss. Despite these challenges, specific antiviral drugs targeting BK virus remain elusive. Agnoprotein, a small, positively charged protein encoded by the BK virus late gene, functions in the assembly, maturation, and release of the virus. Consequently, agnoprotein emerges as a promising target for potential anti-BK virus drugs. Utilizing phage display technology, we conducted screening to identify specific binding peptides against the agnoprotein. The primary objective of screening binding peptides is to utilize them to disrupt the virus’s life cycle, impeding its replication and transmission, thereby achieving antiviral effects. In the current experimental study, a combination of phage 7 peptide libraries and 12 peptide libraries was employed for screening purposes. Following four rounds of screening, seven positive phages demonstrating the ability to bind Agnoprotein were successfully isolated. Following ELISA validation, it was observed that the optical density (OD) values for Agnoprotein binding of the seven positive clones significantly exceeded three times the value of the negative control (NC). Subsequent analysis identified one 7-peptide and six 12-peptides within the binding peptides. Moreover, OD values of dodecapeptide phage clones bound to agnoprotein were generally higher than those of heptapeptide phage clones.In conclusion, our study demonstrates the successful identification of specific binding peptides against agnoprotein, a crucial component in the BK virus life cycle.

Identification of novel KRASG12D neoantigen specific TCRs and a strategy to eliminate off-target recognition

BackgroundT cell receptor (TCR)–engineered T cells targeting neoantigens originated from mutations in KRAS gene have demonstrated promising outcomes in clinical trials against solid tumors. However, the challenge lies in developing tumor-specific TCRs that avoid cross-reactivity with self-antigens to minimize the possibility of severe clinical toxicities. Current research efforts have been put towards strategies to eliminate TCR off-target recognition.MethodsNaive T cell repertoire was used for screening KRASG12D-reactive TCRs. Specific TCRs were subsequently identified and their functionality was assessed using TCR Jurkat cells and TCR T cells. Peptide specificity was evaluated using the X-scan assay. To enhance TCR specificity for KRASG12D and reduce their reactivity to self-peptide SMC1A29-38, mammalian TCR display libraries were employed for the design of modification in the complementarity-determining region (CDR).ResultsHLA-A*11:01-restricted TCRs targeting the KRASG12D epitope were isolated, and TCR1 was characterized with superior functional avidity and specificity. Alongside a robust recognition of endogenous KRASG12D epitope, this TCR displayed cross-reactivity with the SMC1A29-38 epitope. With an approach utilizing structural-guided mutations in the CDR-1A region of TCR1, we obtained an engineered TCR variant (TCR1a7). Functional characterization of TCR1a7 showed that this TCR not only exhibited enhanced specificity towards KRASG12D, but also demonstrated successful elimination of the off-target recognition of SMC1A29-38.ConclusionsTCRs targeting the KRASG12D peptide could be isolated from naive T cell repertoires. Integrating the TCR-peptide-HLA complex structure with a mammalian TCR library system could serve as a functional strategy to reduce potential TCR cross-reactivity with self-antigens, such as SMC1A29-38. Our findings evidenced an operable method to enhance TCR peptide specificity, while maintaining advanced functional avidity and potent anti-tumor activity.

Hybrid lipid nanoparticles with tumor antigen-primed dendritic cell membranes for post-surgical tumor immunotherapy.

Post-surgical tumor recurrence poses a major challenge in cancer treatment due to residual tumor cells and surgery-induced immunosuppression. Here, we developed hybrid nanoparticles, termed T-DCNPs, designed to promote antigen-specific activation of cytotoxic CD8+ T cells while concurrently inhibiting immunosuppressive pathways within the tumor microenvironment. T-DCNPs were formulated by co-extruding lipid nanoparticles containing a transforming growth factor β inhibitor with dendritic cells that were pre-treated with autologous neoantigens derived from surgically excised tumors. By using whole tumor antigens rather than specific peptides, T-DCNPs effectively overcame tumor heterogeneity and elicited a robust, targeted immune response. In vitro studies showed that T-DCNPs enhanced CD8+ T cell proliferation and reduced programmed death-1 (PD-1) expression, leading to increased antitumor cytotoxicity. In vivo experiments, involving intratumoral injections of T-DCNPs in distant tumor and post-surgical melanoma models, demonstrated a significant reduction in distant tumor growth, decreased recurrence rates, and extended survival compared to control groups. Flow cytometry and immunohistochemistry analyses further confirmed the enhanced infiltration of activated CD8+ T cells and a marked reduction in immunosuppressive markers, including PD-1 and Foxp3, within the treated tumors. These results suggest that T-DCNPs, through the dual mechanisms of tumor antigen-specific T cell activation and immune modulation, offer a promising strategy to prevent tumor recurrence following surgery and could potentially improve the efficacy of postoperative cancer immunotherapy.

Dysregulation of Metabolic Peptides in the Gut-Brain Axis Promotes Hyperinsulinemia, Obesity, and Neurodegeneration.

Metabolic peptides can influence metabolic processes and contribute to both inflammatory and/or anti-inflammatory responses. Studies have shown that there are thousands of metabolic peptides, made up of short chains of amino acids, that the human body produces. These peptides are crucial for regulating many different processes like metabolism and cell signaling, as they bind to receptors on various cells. This review will cover the role of three specific metabolic peptides and their roles in hyperinsulinemia, diabetes, inflammation, and neurodegeneration, as well as their roles in type 3 diabetes and dementia. The metabolic peptides glucagon-like peptide 1 (GLP-1), gastric inhibitor polypeptide (GIP), and pancreatic peptide (PP) will be discussed, as dysregulation within their processes can lead to the development of various inflammatory and neurodegenerative diseases. Research has been able to closely investigate the connections between these metabolic peptides and their links to the gut-brain axis, highlighting changes made in the gut that can lead to dysfunction in processes in the brain, as well as changes made in the brain that can lead to dysregulation in the gut. The role of metabolic peptides in the development and potentially reversal of diseases such as obesity, hyperinsulinemia, and type 2 diabetes will also be discussed. Furthermore, we review the potential links between these conditions and neuroinflammation and the development of neurodegenerative diseases like dementia, specifically Parkinson's disease and Alzheimer's disease.

Elucidation of peptide screen for targeted identification of Yersinia pestis by nano-liquid chromatography tandem mass spectrometry

Yersinia pestis, a Gram-negative bacterium is the causative agent of the fatal communicable disease plague. The disease had a profound impact on human history. Plague bacteria are usually transmitted to humans through the bite of an infected rat flea. Earlier studies have indicated that Y. pestis can survive in environmental matrices e.g. water and soil. This study aimed to generate a peptide-based screen for identification of Y. pestis particularly from environmental matrices. We employed a shotgun proteomic approach using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) to discover Y. pestis-specific peptides. The pure cultures of Y. pestis and related species were grown, their proteome were delineated and analyzed by in silico tools to discover 61 Y. pestis specific peptides. Additionally, 148 peptides were discovered from proteins of Y. pestis-specific plasmids and chromosomal-associated virulence markers. To validate this screen of 209 peptides, various concentrations of Y. pestis (ranging from 1.3 × 108 to 1.3 × 105 cfu) were spiked into garden soil. Y. pestis could be identified in all samples except un-spiked negative control soil sample. This study offers a valuable method for the identification of Y. pestis, by tandem mass spectrometry which may be used in environmental and clinical matrices.

The Evolving T Cell Receptor Recognition Code: The Rules Are More Like Guidelines.

αβ T cell receptor (TCR) recognition of peptide-MHC complexes lies at the core of adaptive immunity, balancing specificity and cross-reactivity to facilitate effective antigen discrimination. Early structural studies established basic frameworks helpful for understanding and contextualizing TCR recognition and features such as peptide specificity and MHC restriction. However, the growing TCR structural database and studies launched from structural work continue to reveal exceptions to common assumptions and simplifications derived from earlier work. Here we explore our evolving understanding of TCR recognition, illustrating how structural and biophysical investigations regularly uncover complex phenomena that push against paradigms and expand our understanding of how TCRs bind to and discriminate between peptide/MHC complexes. We discuss the implications of these findings for basic, translational, and predictive immunology, including the challenges in accounting for the inherent adaptability, flexibility, and occasional biophysical sloppiness that characterize TCR recognition.

Recent advances, strategies, and future perspectives of peptide-based drugs in clinical applications.

Peptide-based therapies have attracted considerable interest in the treatment of cancer, diabetes, bacterial infections, and neurodegenerative diseases due to their promising therapeutic properties and enhanced safety profiles. This review provides a comprehensive overview of the major trends in peptide drug discovery and development, emphasizing preclinical strategies aimed at improving peptide stability, specificity, and pharmacokinetic properties. It assesses the current applications and challenges of peptide-based drugs in these diseases, illustrating the pharmaceutical areas where peptide-based drugs demonstrate significant potential. Furthermore, this review analyzes the obstacles that must be overcome in the future, aiming to provide valuable insights and references for the continued advancement of peptide-based drugs.

PEGylated Nanoliposomal Doxorubicin Conjugated with Specific TREM2 Peptides for Glioma-Targeting Therapy.

PEGylated liposomes can deliver anti-cancer drugs to brain tumors, and achieve enhanced permeability and retention effects. Triggering receptor expressed on myeloid cells 2 (TREM2) is an excellent biomarker for precise therapyof glioma. The present study is aimed at designing PEGylatednanoliposomal doxorubicin (PLD) conjugated with peptides targeting TREM2 for glioma-targeting therapy. The specific peptides are designed with the Rosetta Peptiderive Protocol. Schrodinger's peptide-specific version of Glide is used for molecular docking.PLD modified with peptides (peptide-PLD) are engineered and prepared. Cell cycle, apoptosis, cell invasion and migration, cell viability, and colony-formation assays are performed to analyze glioma cell functions. The anti-tumor effects of peptide-PLD are validated in an intracranial U87-MG cells orthotopic glioma model. The targeting peptides HLRKLRKR and LRKLRLRL showed specific affinity for TREM2 and better cellular uptake in U87-MG cells. PLD with peptide modification demonstrated stable doxorubicin loading, small sizes (<60nm), and enrichment in the mouse brain. Peptide-PLD treatment inhibited the Akt/GSK3β/β-catenin pathway, thereby inhibiting cell invasion and migration, and colony-forming ability in U87-MG cells. The peptide modification of PLD achieved better suppression of glioma development than PLD.Overall, TREM2-targeting peptides are successfully designed, and peptide-PLD served as a potent drug delivery carrier for glioma-targeting therapy.

Exploring Multivalency in the Development of Anti-PD-L1 Peptides for Cancer Immunotherapy.

The PD-1/PD-L1 pathway is one of the most effective immune checkpoint pathways utilized for cancer immunotherapy. Despite the success of anti-PD-1/PD-L1 mAbs, there is growing interest in developing low molecular weight anti-PD-1/PD-1 agents, such as peptides, because of their improved tumor penetration. We recently developed a small anti-PD-L1 peptide and demonstrated its promising anti-tumor activity. In this study, we investigate multivalency as a strategy to increase the binding avidity and blocking efficiency of the anti-PD-L1 peptide. Multivalent peptide inhibitors are designed with multiple copies of a peptide inhibitor in a single molecule. We synthesized peptides with different valences and examined their activity. We also investigated how spacer length affects the activity of these multivalent peptides. Using this strategy, we developed a multivalent peptide that demonstrated approximately 40 times higher blocking efficiency and improved stability compared to the original peptide. Increasing the valency enhanced the peptide's specificity, which is essential for minimizing side effects. Multivalency approach represents a promising platform for improving the efficacy of peptide-based checkpoint inhibitors.

Proteomic Sensors for Quantitative, Multiplexed and Spatial Monitoring of Kinase Signaling.

Understanding kinase action requires precise quantitative measurements of their activity in vivo . In addition, the ability to capture spatial information of kinase activity is crucial to deconvolute complex signaling networks, interrogate multifaceted kinase actions, and assess drug effects or genetic perturbations. Here we developed a proteomic kinase activity sensor platform (ProKAS) for the analysis of kinase signaling using mass spectrometry. ProKAS is based on a tandem array of peptide sensors with amino acid barcodes that allow multiplexed analysis for spatial, kinetic, and screening applications. We engineered a ProKAS module to simultaneously monitor the activities of the DNA damage response kinases ATR, ATM, and CHK1 in response to genotoxic drugs, while also uncovering differences between these signaling responses in the nucleus, cytosol, and replication factories. Furthermore, we developed an in silico approach for the rational design of specific substrate peptides expandable to other kinases. Overall, ProKAS is a novel versatile system for systematically and spatially probing kinase action in cells.

Co-incubation of Short Amphiphilic Peptides with Dicer Substrate RNAs Results in β-Sheet Fibrils for Enhanced Gene Silencing in Cancer Cells

RNA can interact with positively charged, amphiphilic peptides to cooperatively assemble into fibrils that enable RNA transport across cancer cellular membranes. RNA decreases the folding energy barrier imposed by the electrostatic repulsion between these charged peptides, thus partaking in RNA-peptide self-assembly along particular pathways in the energy landscape. Specific amphiphilic peptides capable of protecting and transporting RNA across a membrane have Type II’ β-turn hairpin forming motifs in their structures, which aids self-assembly into β-sheet fibrils. We employed a set of such cationic, amphiphilic peptides that have random coiled structures in the absence of folding stimuli, to characterize the (peptides):(RNA) assembly. We subjected these complexes to extensive biophysical characterization in vitro and in cell culture. We show that short RNAs (such as Dicer substrate RNAs) can lead these peptides to self-assemble into β-sheet fibrils that have RNA transport capabilities and can act as non-viral delivery vectors for RNA. Modulation in the peptide sequence implicitly alters the way they bind RNA and influence the peptides’ ability to transport nucleic acids across membranes.

Quantitative detection of the maize phytocytokine Zip1 utilizing ELISA.

Plant signaling peptides, also known as phytocytokines, play a crucial role in cell-to-cell communication during plant development and immunity. The detection of small peptides in plant tissues is challenging and often relies on time-consuming and cost-intensive approaches. Here, we present an ELISA-based assay as a rapid and cost-effective method for the detection of naturally released peptides in plant tissues. Our ELISA-based method was developed to detect Zip1, a 17-amino-acid phytocytokine derived from Zea mays that elicits salicylic acid signaling in maize leaves. Using a custom peptide-antibody, we designed an experimental pipeline to achieve peptide specificity, selectivity, and sensitivity allowing the detection of the Zip1 peptide in complex biological samples. As a proof of concept, we first overexpressed the precursor molecule PROZIP1 in Nicotiana benthamiana and in transfected maize protoplasts and monitored the release of Zip1-containing peptides. In a second approach we treated maize leaves with salicylic acid to induce native PROZIP1 expression and processing. Using ELISA, we were able to quantify native Zip1 signals with a detection limit in the nanogram range, which allowed us to detect different Zip1-containing peptides in plant material. This method can be adapted for the detection and quantification of a variety of plant signaling peptides.

FVIII peptides presented on HLA-DP and identification of an A3 domain peptide binding with high affinity to the commonly expressed HLA-DP4.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

Characterization of VldE (Spr1875), a Pneumococcal Two-State l,d-Endopeptidase with a Four-Zinc Cluster in the Active Site.

Remodeling of the pneumococcal cell wall, carried out by peptidoglycan (PG) hydrolases, is imperative for maintaining bacterial cell shape and ensuring survival, particularly during cell division or stress response. The Streptococcus pneumoniae protein Spr1875 plays a role in stress response, both regulated by the VicRK two-component system (analogous to the WalRK TCS found in Firmicutes). Modular Spr1875 presents a putative cell-wall binding module at the N-terminus and a catalytic C-terminal module (Spr1875MT3) connected by a long linker. Assays of the full-length protein and Spr1875MT3 with PG-based synthetic substrates by liquid chromatography/mass spectrometry revealed Spr1875 as an l,d-endopeptidase, renamed VldE (for VicRK-regulated l,d-endopeptidase), which hydrolyzed the cross-linked stem peptide in the PG. Remarkably, we observed asymmetric turnover with specific recognition of the acceptor peptide strand. Localization experiments showed that the protein is directed to the septum, which suggests that muralytic activity could be required for pneumococcal growth under stress conditions. Our findings, based on six high-resolution X-ray crystallographic structures and molecular-dynamics simulations, reveal two states for VldEMT3. The protein transitions between a noncatalytic state that binds up to four zinc ions, thus behaving as a Zn2+ reservoir, and a catalytic state that performs the hydrolytic reaction with a single zinc ion. Furthermore, computational studies provide insight into the mechanism of catalytic-water activation and nucleophilic attack on the specific scissile peptide bond of the asymmetric cross-linked PG.