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Bone diseases are medical conditions caused by the loss of bone homeostasis consecutive to increased osteoclast activity and diminished osteoblast activity. The mevalonate pathway (MVA) is crucial for maintaining this balance since it drives the post-translational prenylation of small guanosine triphosphatases (GTPases) proteins. Farnesyl pyrophosphate synthase (FPPS) plays a crucial role in the MVA pathway. Consequently, in the treatment of bone-related diseases, FPPS is the target of FDA-approved nitrogen-containing bisphosphonates (N-BPs), which have tropism mainly for bone tissue due to their poor penetration in soft tissues. The development of inhibitors targeting the FPPS enzyme has garnered significant interest in recent decades due to FPPS's role in the biosynthesis of cholesterol and other isoprenoids, which are implicated in cancer, bone diseases, and other conditions. In this study, we describe a multidisciplinary approach to designing novel FPPS inhibitors, combining computational modeling, biochemical assays, and biophysical techniques. A series of peptides and phosphopeptides were designed, synthesized, and evaluated for their ability to inhibit FPPS activity. Molecular docking was employed to predict the binding modes of these compounds to FPPS, while Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) spectroscopy experiments - based on Saturation Transfer Difference (STD) and an enzymatic NMR assay - were used to measure their binding affinities and kinetics. The biological activity of the most promising compounds was further assessed in cellular assays using murine colorectal cancer (CRC) cells. Additionally, genomics and metabolomics profiling allowed to unravel the possible mechanisms underlying the activity of the peptides, confirming their involvement in the modulation of the MVA pathway. Our findings demonstrate that the designed peptides and phosphopeptides exhibit significant inhibitory activity against FPPS and possess antiproliferative effects on CRC cells, suggesting their potential as therapeutic agents for cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by extracellular acidification. Amongst many pathological roles, ASICs are an important mediator of ischemic cell death and hence an attractive drug target for stroke treatment as well as other conditions. A peptide called Hi1a, isolated from Australian funnel web spider venom, inhibits ASIC1a and attenuates cell death in a stroke model up to 8 h after stroke induction. Here, we set out to understand the molecular basis for Hi1a's action. Hi1a is a bivalent toxin with two inhibitory cystine knot domains joined by a short linker. We found that both Hi1a domains modulate human ASIC1a gating with the N-terminal domain impairing channel activation while the C-terminal domain produces a "pro-open" phenotype even at submicromolar concentrations. Interestingly, both domains bind at the same site since a single point mutation, F352A, abolishes functional effects and reduces toxin affinity in surface plasmon resonance measurements. Therefore, the action of Hi1a at ASIC1a appears to arise through a mutually exclusive binding model where either the N or C domain of a single Hi1a binds one ASIC1a subunit. An ASIC1a trimer may bind several inhibitory N domains and one or more pro-open C domains at any one time, accounting for the incomplete inhibition of wild type Hi1a. We also found that the functional differences between these two domains are partially transferred by mutagenesis, affording new insight into the channel function and possible novel avenues of drug design., (© 2024 Berger and MacLean.)

We previously reported that a bioactive peptide (pep3) can potently inhibit the enzyme activity of purified calcineurin (CN). In this paper, we further demonstrate that transfected pep3 can strongly inhibit CN enzyme activity in HEK293 cells. Transcription factor EB (TFEB) plays an important role in the autophagy-lysosome pathway (ALP) as one of the substrates of CN, so we study the effect of pep3 on the CN-TFEB-ALP pathway. Pep3 can significantly inhibit the mRNA levels of the TFEB downstream genes and the expression of the autophagy-associated proteins, and autophagy flux in HEK293 cells. We also validated the inhibitory effect of pep3 on autophagy in mice. These findings may provide a new idea for discovering more CN inhibitors and autophagy inhibitory drugs., Competing Interests: Declaration of competing interest We confirm that this work is original and not written by AI, nor has it been published elsewhere. All authors have no conflicts of interest that need to be disclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In eukaryotes, gene expression typically requires individual promoter and terminator for each gene, making the expression of multiple genes tedious and sometimes too difficult to handle. This is especially true for underdeveloped nonmodel organisms with few genetic engineering tools and genetic elements such as Rhodosporidium toruloides. In contrast, polycistronic expression offers advantages such as smaller size and ease of cloning. Here we report the development of a multigene expression system using 2A peptides in R. toruloides. First, twenty-two 2A peptides were evaluated for their cleavage efficiencies, which ranged from 33.65% to 93.32%. Subsequently, the 2A peptide of ERBV-1 with the highest efficiency was selected to enable simultaneous expression of four proteins. In addition, we demonstrated the optimization of the α-linolenic acid biosynthetic pathway using ERBV-1 peptide mediated polycistronic expression, which increased the α-linolenic acid production by 104.72%. These results suggest that using ERBV-1 peptide is an efficient strategy for multigene expression in R. toruloides., (© 2024 Wiley Periodicals LLC.)

Soil Pb contamination is inevitable, as a result of phosphate mining. It is essential to explore more effective Pb remediation approaches in phosphate mining wasteland soil to ensure their viability for a gradual return of soil quality for cultivation. In this study, a Pb-resistant urease-producing bacterium, Serratia marcescens W1Z1, was screened for remediation using microbially induced carbonate precipitation (MICP). Magnesium polypeptide (MP) was prepared from soybean meal residue, and the combined remediation of Pb contamination with MP and MICP in phosphate mining wasteland soil was studied. Remediation of Pb using a combination of MP with MICP strain W1Z1 (WM treatment) was the most effective, with the least exchangeable Pb at 30.37% and the most carbonate-bound Pb at 40.82%, compared to the other treatments, with a pH increase of 8.38. According to the community analysis, MP moderated the damage to microbial abundance and diversity caused by MICP. Total nitrogen (TN) was positively correlated with Firmicutes, pH, and carbonate-bound Pb. Serratia inoculated with strain W1Z1 were positively correlated with bacteria belonging to the Firmicutes phylum and negatively correlated with bacteria belonging to Proteobacteria. The available phosphate (AP) in the phosphate mining wasteland soil could encapsulate the precipitated Pb by ion exchange with carbonate, making it more stable. Combined MP-MICP remediation of Pb contamination in phosphate mining wasteland soil was effective and improved the soil microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Medin amyloid, prevalent in the vessel walls of 97 % of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin 1-10 and medin 11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin 31-40 exhibited moderate aggregation, forming dynamic β-sheet oligomers with frequent association and dissociation. Conversely, medin 21-30 and medin 41-50 segments demonstrated significant self-assembly capability, readily forming stable β-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin 21-30 and medin 41-50 , acting as the β-sheet core and facilitating β-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in β-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Dravet syndrome is a rare form of epilepsy starting from infancy that can plaque the affected individuals all though his/her life with repeated seizures, and this condition is currently without a complete cure. So prenatal screening of molecular markers of this condition is urgently needed to help couples conceiving new lives to steer clear of this potential danger. And such an assay should ideally be of low cost and could be completed in a point-of-care fashion. This work reports an attempt to construct such an assay using simple peptides in the place of conventional biosensing macro-molecules such as antibodies and enzymes. Specifically, a marker protein of this syndrome can bring the two pieces of a self-splitting peptide "intein" together, which in turn facilitate the formation of metal ion coordination site, recruiting cupric ion to generate catalytically amplified signal readout. Using this method, disease marker protein Nav of this syndrome can be quantitatively detected directly in amniotic fluid samples, and samples associated with potential risk factors such as family history of this syndrome shows statistically evident decrease of this marker protein. These results may promise future application of the proposed method in clinical practice to reduce the social burden of Dravet syndrome by reducing its actual incident rate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Inhibitory activity against angiotensin-converting enzyme (IAACE) by chicken skin collagen hydrolysate (CSCH) and their peptide fractions before and after in-vitro gastrointestinal digestion, were evaluated; as well as their ability to modulate lipid accumulation in 3 T3-L1 adipocytes. Before digestion, peptide fraction <1 kDa (F4) showed the highest IAACE (p < 0.05) followed by CSCH. After these samples were digested, F4 presented an IAACE with IC 50 similar to its digest (DF4) (188.84 and 220.03 μg/mL, respectively), which was 2-fold lower (p < 0.05) than IC 50 of fraction <1 kDa from post-digested hydrolysate (FDH) (388.57 μg/mL). Nine peptides were identified as the potential ACE inhibitors in F4 and DF4. Addition of DF4 (800 μg/mL) reduced(p < 0.05) lipid accumulation by 83% within preadipocytes. A 45-60% reduction of lipid accumulation within differentiated adipocytes was obtained by adding FDH and DF4 (regardless the concentration). These results, digested CSCH and F4 with IAACE may be considered as potential adjuvants for obesity treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Cheesemaking with camel milk (CM) presents unique challenges and additional health benefits. This study involved preparing low-fat Cheddar cheese (LFCC) by blending bovine milk (BM) with varying levels of CM. Control cheese was made exclusively with BM. After 180 days of ripening, LFCC samples underwent in vitro digestion to determine antioxidant capacities, α-amylase and α-glucosidase inhibition, and angiotensin-converting enzyme inhibition. The peptide profile of LFCC treatments was analyzed using liquid chromatography-quadrupole-time of flight-mass spectrometry. Antioxidant and biological activities were influenced by BM-CM blends and digestion. At days 120 and 180, the number of αs1-casein-derived peptides increased in all samples except for LFCC made with 15% CM. Generally, 88 peptides exhibited ACE inhibition activity after 120 days of ripening, increasing to 114 by day 180. These findings suggest that ripening time positively affects the health-promoting aspects of functional cheese products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

During infection, the host employs nutritional immunity to restrict access to iron. Staphylococcus lugdunensis has been recognized for its ability to utilize host-derived heme to overcome iron restriction. However, the mechanism behind this process involves the release of hemoglobin from erythrocytes, and the hemolytic factors of S. lugdunensis remain poorly understood. S. lugdunensis encodes four phenol-soluble modulins (PSMs), short peptides with hemolytic activity. The peptides SLUSH A, SLUSH B, and SLUSH C are β-type PSMs, and OrfX is an α-type PSM. Our study shows the SLUSH locus to be essential for the hemolytic phenotype of S. lugdunensis. All four peptides individually exhibited hemolytic activity against human and sheep erythrocytes, but synergism with sphingomyelinase was observed exclusively against sheep erythrocytes. Furthermore, our findings demonstrate that SLUSH is crucial for allowing the utilization of erythrocytes as the sole source of nutritional iron and confirm the transcriptional regulation of SLUSH by Agr. Additionally, our study reveals that SLUSH peptides stimulate the human immune system. Our analysis identifies SLUSH as a pivotal hemolytic factor of S. lugdunensis and demonstrates its concerted action with heme acquisition systems to overcome iron limitation in the presence of host erythrocytes., (© 2024 Federation of American Societies for Experimental Biology.)

This study aimed to investigate the antioxidative and cytoprotective activity of antioxidant peptides from fermented broad bean paste (FBBP) and explore their potential molecular mechanisms using a combined in silico and in vitro approach. Seven novel antioxidant peptides (VSRRFIYYL, SPAIPLP, PVPPPGG, KKDGYWWAKFK, LAWY, LGFMQF, and LPGCP) identified by integrated approaches of peptidomics and in silico bioinformatic analysis were synthesized, exhibiting strong antioxidant potential against in vitro radicals. Molecular docking results suggested that these peptides could form stable hydrogen bonds and solvent-accessible surface with key amino acid residues of Keap1, thus potentially regulating the Keap1-Nrf2 pathway by occupying the Nrf2-binding site on Keap1. Additionally, they exhibited strong cellular antioxidant activity and could protect HepG2 cells from AAPH-induced oxidative injury by reducing reactive oxygen species and MDA accumulation. This study firstly unraveled the molecular mechanisms of antioxidant peptides from FBBP, and provided a new theoretical basis for the high-value utilization of FBBP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA's self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43 Tg -ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy., (© 2024. The Author(s).)

Amid the growing concern for health-oriented food choices, salt reduction has received widespread attention, particularly in the exploitation of salt alternatives. Peptides with a saltiness-enhancing effect may provide an alternative method for salt reduction. The objective of this study was to isolate and extract novel peptides with salt-reducing effects by fermenting goose blood using a Lactobacillus plantarum strain. Five potential target peptides were screened by a virtual database prediction and molecular docking. Sensory evaluation and E-tongue analysis showed that five peptides (NEALQRM, GDAVKNLD, HAYNLRVD, PEMHAAFDK, and AEEKQLITGL) were identified as target peptides. Particularly, the results of E-tongue showed that GDAVKNLD can increase the saltiness intensity (2.87 ± 0.02) in the complex system. The sensory evaluation results also indicated an increase in saltiness intensity (46.67 ± 4.67 mmol/L NaCl) after adding GDAVKNLD. The results of molecular dynamics simulation indicated that five peptides have good ability to bind tightly to TMC4 receptor, thereby stimulating it to exert an active effect. And these peptides interacted with the TMC4 receptor via hydrogen bonding, hydrophobic interactions, and electrostatic interactions. This research lays a theoretical foundation for discovering novel salty/saltiness-enhancing peptides and provides meaningful contributions to efforts in salt reduction.

In the brewing process, methionine is a decisive amino acid for (off-)flavor formation. A significant part of methionine is oxidized to methionine sulfoxide (MetSO) in malt. We hypothesized that MetSO and MetSO 2 are metabolized to volatile compounds during yeast fermentation and examined whether the yeast Saccharomyces cerevisiae is able to catabolize l-MetSO and l-MetSO 2 in free and dipeptide-bound forms. We also investigated the stability of l-methionine sulfoximine and S -methylmethionine. Cell viability in the presence of the test compounds was at least 90%. Both free and peptide-bound test substances were metabolized by Saccharomyces cerevisiae . l-MetSO was degraded most rapidly as the free amino acid, while l-MetSO 2 was degraded most rapidly bound in dipeptides. We observed a different degradation behavior of the ( R ) and ( S ) diastereoisomers for l-MetSO and l-methionine sulfoximine. Furthermore, we detected methionol as the only metabolite of MetSO. Methionol sulfoxide was not formed. MetSO 2 was not converted to methionol or methionol sulfone but to the respective α-hydroxy acid. We conclude that the reduction of MetSO to methionine proceeds faster than transamination. The occurrence of MetSO or MetSO 2 in brewing malt will not lead to the formation of hitherto unknown volatile metabolites of the Ehrlich pathway.

Amidst increasing awareness of diet-health relationships, plant-derived bioactive peptides are recognized for their dual nutritional and health benefits. This study investigates bioactive peptides released after Alcalase hydrolysis of protein from chachafruto ( Erythrina edulis ), a nutrient-rich South American leguminous plant, focusing on their behavior during simulated gastrointestinal digestion. Evaluating their ability to scavenge radicals, mitigate oxidative stress, and influence immune response biomarkers, this study underscores the importance of understanding peptide interactions in digestion. The greatest contribution to the antioxidant activity was exerted by the low molecular weight peptides with ORAC values for the <3 kDa fraction of HES, GD-HES, and GID-HES of 0.74 ± 0.03, 0.72 ± 0.004, and 0.56 ± 0.01 (μmol TE/mg protein, respectively). GD-HES and GID-HES exhibited immunomodulatory effects, promoting the release of NO up to 18.52 and 8.58 µM, respectively. The findings of this study highlighted the potential of chachafruto bioactive peptides in functional foods and nutraceuticals, supporting human health through dietary interventions.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

With the rapid development of the medical beauty industry, functional skin care products become increasingly popular. The functions of cosmetics mainly depend on the active ingredients, which are mainly proteins, peptides, polysaccharides, phenolic acids, terpenes, vitamins, and amino acids. These active ingredients endow cosmetics with skin repairing, moistening, whitening, UV protecting, and anti-aging effects. They are mainly obtained through biological extraction and chemical synthesis. In recent years, with the development of biomanufacturing, microbial synthesis of active ingredients in cosmetics has been widely studied and applied. This article reviews the research progresses in the production of natural products including collagens, peptides, hyaluronic acid, polyphenols, terpenes, and vitamins by microbial synthetic biotechnology. Moreover, this article highlighted the synthetic pathways, metabolic regulation, and prospects of the natural products, providing a reference for subsequent microbial synthesis of active ingredients in cosmetics.

Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery., (© 2024. The Author(s).)

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvβ3., (© 2024. The Author(s).)

Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but are impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions., (© 2024. The Author(s).)

C5a is an integral glycoprotein of the complement system that plays an important role in inflammation and immunity. The physiological concentration of C5a is observed to be elevated under various immunoinflammatory pathophysiological conditions in humans. The pathophysiology of C5a is linked to the "two-site" protein-protein interactions (PPIs) with two genomically related receptors, such as C5aR1 and C5aR2. Therefore, pharmacophores that can potentially block the PPIs between C5a-C5aR1 and C5a-C5aR2 have tremendous potential for development as future therapeutics. Notably, the FDA has already approved antibodies that target the precursors of C5a (Eculizumab, 148 kDa) and C5a (Vilobelimab, 149 kDa) for marketing as complement-targeted therapeutics. In this context, the current study reports the structural characterization of a pair of synthetic designer antibody-like peptides (DePA and DePA1; ≤3.8 kDa) that bind to hotspot regions on C5a and also demonstrates potential traits to neutralize the function of C5a under pathophysiological conditions.

A variety of therapeutic possibilities have emerged for skillfully regulating protein function or conformation through intermolecular interaction modulation to rectify abnormal biochemical reactions in diseases. Herein, a devised strategy of enzyme coordinators has been employed to alleviate postoperative pancreatic fistula (POPF), which is characterized by the leakage of digestive enzymes including trypsin, chymotrypsin, and lipase. The development of a dextrorotary (D)-peptide supramolecular gel (CP-CNDS) under this notion showcases its propensity for forming gels driven by intermolecular interaction. Upon POPF, CP-CNDS not only captures enzymes from solution into hydrogel, but also effectively entraps them within the internal gel, preventing their exchange with counterparts in the external milieu. As a result, CP-CNDS completely suppresses the activity of digestive enzymes, effectively alleviating POPF. Remarkably, rats with POPF treated with CP-CNDS not only survived but also made a recovery within a mere 3-day period, while mock-treated POPF rats had a survival rate of less than 5 days when experiencing postoperative pancreatic fistula, leak or abscess. Collectively, the reported CP-CNDS provides promising avenues for preventing and treating POPF, while exemplifying precision medicine-guided regulation of protein activity that effectively targets specific pathogenic molecules across multiple diseases., (© 2024. The Author(s).)

While the cellular cytosol and organelles contain attractive targets for disease treatments, it remains a challenge to deliver therapeutic biomacromolecules to these sites. This is due to the selective permeability of the plasma and endosomal membranes, especially for large and hydrophilic therapeutic cargos such as proteins and nucleic acids. In response, many different delivery systems and molecules have been devised to help therapeutics cross these barriers to reach cytosolic targets. Among them are peptide and protein-based systems, which have several advantages over other natural and synthetic materials including their ability to interact with cell membranes. In this review, we will describe recent advances and current challenges of peptide and protein strategies that leverage cell membrane association and modulation to enable cytosolic delivery of biomacromolecule cargo. The approaches covered here include peptides and proteins derived from or inspired by natural sequences as well as those designed de novo for delivery function.

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.

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG) 3 . Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Replacing cereals with food leftovers could reduce feed-food competition and keep nutrients and energy in the food chain. Former food products (FFPs) are industrial food leftovers no more intended for human but still suitable as alternative and sustainable feedstuffs for monogastric. In this study, omics approaches were applied to evaluate the impact of dietary FFPs on pig liver proteome and plasma peptidome. Thirty-six Swiss Large White male castrated pigs were randomly assigned to three dietary treatments [control (CTR), 30% CTR replaced with salty FFP (SA), 30% CTR replaced with sugary FFP (SU)] from the start of the growing phase (22.4 ± 1.7 kg) until slaughtering (110 ± 3 kg). The low number of differentially regulated proteins in each comparison matrix (SA/SU vs. CTR) and the lack of metabolic interaction indicated a marginal impact on hepatic lipid metabolism. The plasma peptidomics investigation showed low variability between the peptidome of the three dietary groups and identified three possible bioactive peptides in the SA group associated with anti-hypertension and vascular homeostasis regulation. To conclude, the limited modulation of liver proteome and plasma peptidome by the SA and SU diets strenghtened the idea of reusing FFPs as feed ingredients to make pig production more sustainable., (© 2024. The Author(s).)

Recent advances in mass spectrometry-based peptidomics have catalyzed the identification and quantification of thousands of endogenous peptides across diverse biological systems. However, the vast peptidomic landscape generated by proteolytic processing poses several challenges for downstream analyses and limits the comparability of clinical samples. Here, we present an algorithm that aggregates peptides into peptide clusters, reducing the dimensionality of peptidomics data, improving the definition of protease cut sites, enhancing inter-sample comparability, and enabling the implementation of large-scale data analysis methods akin to those employed in other omics fields. We showcase the algorithm by performing large-scale quantitative analysis of wound fluid peptidomes of highly defined porcine wound infections and human clinical non-healing wounds. This revealed signature phenotype-specific peptide regions and proteolytic activity at the earliest stages of bacterial colonization. We validated the method on the urinary peptidome of type 1 diabetics which revealed potential subgroups and improved classification accuracy., (© 2024. The Author(s).)

MPIase is a glycolipid involved in protein insertion into and preprotein translocation across the cytoplasmic membranes of E. coli. MPIase is upregulated in the cold conditions to overcome the cold-sensitive protein export. CdsA, a CDP-diacylglycerol synthase, catalyzes the first reaction in MPIase biosynthesis. An open reading frame for a peptide of 50 amino acids is encoded immediately after ispU, a neighboring upstream gene of cdsA, and overlaps cdsA to a large extent. Mutational analysis revealed that the expression of this peptide is essential for upregulation of MPIase in the cold. Consistently, expression of this peptide in trans resulted in cold upregulation of MPIase. We therefore named this peptide MucA after its function (MPIase upregulation in the cold). When the partially purified MucA was added to the reaction of the intermediate in MPIase biosynthesis, a significant increase in the product formation was observed, supporting the function of MucA. The possible role of MucA in MPIase biosynthesis is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with regard to the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The kallikrein-related peptidase KLK2 has restricted expression in the prostate luminal epithelium, and its protein target is unknown. The present work reports the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy-transfer peptides from which the sequence SYRIF was the most susceptible substrate for KLK2. The sequence SYRIF is present at the extracellular N -terminal segment ( 58 SYRIF 63 Q) of IL-10R2. KLK2 was fully active at pH 8.0-8.2, found only in prostate inflammatory conditions, and strongly activated by sodium citrate and glycosaminoglycans, the quantities and structures controlled by prostate cells. Bone-marrow-derived macrophages (BMDM) have IL-10R2 expressed on the cell surface, which is significantly reduced after KLK2 treatment, as determined by flow cytometry (FACS analysis). The IL-10 inhibition of the inflammatory response to LPS/IFN-γ in BMDM cells due to decreased nitric oxide, TNF-α, and IL-12 p40 levels is significantly reduced upon treatment of these cells with KLK2. Similar experiments with KLK3 did not show these effects. These observations indicate that KLK2 proteolytic activity plays a role in prostate inflammation and makes KLK2 a promising target for prostatitis treatment.

Naturally occurring lanthipeptides, peptides post-translationally modified by various enzymes, hold significant promise as antibiotics. Despite extensive biochemical and structural studies, the events preceding peptide modification remain poorly understood. Here, we identify a distinct subclass of lanthionine synthetase KC (LanKC) enzymes with distinct structural and functional characteristics. We show that PneKC, a member of this subclass, forms a dimer and possesses GTPase activity. Through three cryo-EM structures of PneKC, we illustrate different stages of peptide PneA binding, from initial recognition to full binding. Our structures show the kinase domain complexed with the PneA core peptide and GTPγS, a phosphate-bound lyase domain, and an unconventional cyclase domain. The leader peptide of PneA interact with a gate loop, transitioning from an extended to a helical conformation. We identify a dimerization hot spot and propose a "negative cooperativity" mechanism toggling the enzyme between tense and relaxed conformation. Additionally, we identify an important salt bridge in the cyclase domain, differing from those in in conventional cyclase domains. These residues are highly conserved in the LanKC subclass and are part of two signature motifs. These results unveil potential differences in lanthipeptide modification enzymes assembly and deepen our understanding of allostery in these multifunctional enzymes., (© 2024. The Author(s).)

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β 2 -adrenergic receptor (β 2 AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB 7 , Car 9 , and Si 4-1 ) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β 2 -adrenoceptor (β 2 AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β 2 AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The K A s of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β 2 AR-CotB1p column were 1.26 × 10 6 , 6.59 × 10 6 , 7.90 × 10 6 , and 8.97 × 10 5 M -1 respectively, which were two orders of magnitude higher than those on the Halo-β 2 AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Huntington's disease (HD) is a fatal neurodegenerative disorder resulting from an abnormal expansion of polyglutamine (polyQ) repeats in the N-terminus of the huntingtin protein. When the polyQ tract surpasses 35 repeats, the mutated protein undergoes misfolding, culminating in the formation of intracellular aggregates. Research in mouse models suggests that HD pathogenesis involves the aggregation of N-terminal fragments of the huntingtin protein (htt). These early oligomeric assemblies of htt, exhibiting diverse characteristics during aggregation, are implicated as potential toxic entities in HD. However, a consensus on their specific structures remains elusive. Understanding the heterogeneous nature of htt oligomers provides crucial insights into disease mechanisms, emphasizing the need to identify various oligomeric conformations as potential therapeutic targets. Employing coarse-grained molecular dynamics, our study aims to elucidate the mechanisms governing the aggregation process and resultant aggregate architectures of htt. The polyQ tract within htt is flanked by two regions: an N-terminal domain (N17) and a short C-terminal proline-rich segment. We conducted self-assembly simulations involving five distinct N17 + polyQ systems with polyQ lengths ranging from 7 to 45, utilizing the ProMPT force field. Prolongation of the polyQ domain correlates with an increase in β-sheet-rich structures. Longer polyQ lengths favor intramolecular β-sheets over intermolecular interactions due to the folding of the elongated polyQ domain into hairpin-rich conformations. Importantly, variations in polyQ length significantly influence resulting oligomeric structures. Shorter polyQ domains lead to N17 domain aggregation, forming a hydrophobic core, while longer polyQ lengths introduce a competition between N17 hydrophobic interactions and polyQ polar interactions, resulting in densely packed polyQ cores with outwardly distributed N17 domains. Additionally, at extended polyQ lengths, we observe distinct oligomeric conformations with varying degrees of N17 bundling. These findings can help explain the toxic gain-of-function that htt with expanded polyQ acquires.

This study aimed to identify saltiness-enhancing peptides from yeast protein and elucidate their mechanisms by molecular docking. Yeast protein hydrolysates with optimal saltiness-enhancing effects were prepared under conditions determined using an orthogonal test. Ten saltiness-enhancing peptide candidates were screened using an integrated virtual screening strategy. Sensory evaluation demonstrated that these peptides exhibited diverse taste characteristics (detection thresholds: 0.13-0.50 mmol/L). Peptides NKF, LGLR, WDL, NMKF, FDSL and FDGK synergistically or additively enhanced the saltiness of a 0.30% NaCl solution. Molecular docking revealed that these peptides predominantly interacted with TMC4 by hydrogen bonding, with hydrophilic amino acids from both peptides and TMC4 playing a pivotal role in their binding. Furthermore, Leu217, Gln377, Glu378, Pro474 and Cys475 were postulated as the key binding sites of TMC4. These findings establish a robust theoretical foundation for salt reduction strategies in food and provide novel insights into the potential applications of yeast proteins., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The nascent polypeptide-associate complex (NAC) is a heterodimeric chaperone complex that binds near the ribosome exit tunnel and is the first point of chaperone contact for newly synthesized proteins. Deletion of the NAC induces embryonic lethality in many multi-cellular organisms. Previous work has shown that the deletion of the NAC rescues cells from prion-induced cytotoxicity. This counterintuitive result led us to hypothesize that NAC disruption would improve viability in cells expressing human misfolding proteins. Here, we show that NAC disruption improves viability in cells expressing expanded polyglutamine and also leads to delayed and reduced aggregation of expanded polyglutamine and changes in polyglutamine aggregate morphology. Moreover, we show that NAC disruption leads to changes in de novo yeast prion induction. These results indicate that the NAC plays a critical role in aggregate organization as a potential therapeutic target in neurodegenerative disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dublin-Ryan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 3 10 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Joost Snijder reports financial support was provided by Dutch Research Council. Bert Janssen reports financial support was provided by Dutch Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Zearalenone (ZEN) is an estrogenic mycotoxin causing reproductive toxicity in livestock. Currently, lactone hydrolases are used in the enzymatic degradation of ZEN. However, most lactone hydrolases suffer from low degradation efficiency and poor thermal stability. ZHD518, as a documented neutral enzyme for ZEN degradation, exhibits high enzymatic activity under neutral conditions. In this study, a multifunctional peptide S1v1-(AEAEAHAH) 2 was fused to the N-terminus of ZHD518. Compared with the wild-type enzyme, the peptide fusion significantly enhanced protein expression by 1.28 times, enzyme activity by 9.27 times, thermal stability by 37.08 times after incubation at 45 °C for 10 min and enzyme stability during long-term storage. Moreover, ZEN concentrations in corn bran, corn germ meal, and corn gluten powder decreased from 5.29 ± 0.04, 5.31 ± 0.03, and 5.30 ± 0.01 μg/g to 0.48 ± 0.05, 0.48 ± 0.06, and 0.21 ± 0.04 μg/g, respectively, following a 60 min treatment with S1v1-GS-ZHD518, resulting in degradation rates of 90.98, 91.00, and 95.32%, respectively. In conclusion, the properties of S1v1-GS-ZHD518, such as its efficient degradability, high temperature resistance and storage resistance, offer the possibility of its application in food or feed.

In this study, the transepithelial transport of bioactive peptides derived from faba bean flour gastrointestinal digestates was investigated, in vitro, using a Caco-2 and HT29-MTX-E12 coculture monolayer, in comparison to those of pea and soy. The profile of transported peptides was determined by mass spectrometry, and the residual antioxidant activity was assessed. The ORAC value significantly ( p < 0.05) decreased after transepithelial transport (24-36% reduction) for all legumes, while the antioxidant activity in ABTS assay significantly ( p < 0.05) increased, as shown by the EC 50 decrease of 26-44%. Five of the nine faba bean peptides that crossed the intestinal cell monolayer exhibited antioxidant activity. Two of these peptides, TETWNPNHPEL and TETWNPNHPE, were further hydrolyzed by the cells' brush border peptidases to smaller fragments TETWNPNHP and TWNPNHPE. These metabolized peptides were synthesized, and both maintained high antioxidant activity in both ABTS (EC 50 of 1.2 ± 0.2 and 0.4 ± 0.1 mM, respectively) and ORAC (2.5 ± 0.1 and 3.4 ± 0.2 mM of Trolox equivalent/mM, respectively) assays. These results demonstrated for the first time the bioaccessibility of faba bean peptides produced after in vitro gastrointestinal digestion and how their bioactive properties can be modulated during transepithelial transport.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".

Inspired by natural metallopeptides, our work focuses on engineering self-assembling nanostructures of C 2 -symmetric metallopeptide conjugates (MPC) from a pyridine-bis-tripeptide bioprobe that uniquely detects lead (Pb 2+ ) ions by emitting a fluorescence signal at 450 nm, which is further intensified in the presence of DAPI ( λ em = 458 nm), enhancing the bioimaging quality. This study enables precise lead quantification by modulating the ionic conformation and morphology. Experimental and theoretical insights elucidate the nanostructure formation mechanism, laying the groundwork for materials encapsulation and advancing lead detoxification. Our proof-of-principle experiment, demonstrating actin filament recovery in lead-treated cells, signifies therapeutic potential for intracellular lead aggregation and introduces novel avenues in biotechnological applications within biomaterials science.

Hepatocellular carcinoma (HCC) still presents poor prognosis with low overall survival rates and limited therapeutic options available. Recently, attention has been drawn to peptidomic analysis, an emerging field of proteomics for the exploration of new potential peptide drugs for the treatment of various diseases. However, research on the potential function of HCC peptides is lacking. Here, we analyzed the peptide spectrum in HCC tissues using peptidomic techniques and explored the potentially beneficial peptides involved in HCC. Changes in peptide profiles in HCC were examined using liquid chromatography-mass spectrometry (LC-MS/MS). Analyze the physicochemical properties and function of differently expressed peptides using bioinformatics. The effect of candidate functional peptides on HCC cell growth and migration was evaluated using the CCK-8, colony formation, and transwell assays. Transcriptome sequencing analysis and western blot were employed to delve into the mode of action of potential peptide on HCC. Peptidomic analysis of HCC tissue yielded a total of 8683 peptides, of which 452 exhibited up-regulation and 362 showed down-regulation. The peptides that were differentially expressed, according to bioinformatic analysis, were closely linked to carbon metabolism and the mitochondrial inner membrane. The peptide functional validation identified a novel peptide, PDLC (peptide derived from liver cancer), which was found to dramatically boost HCC cell proliferation through the Ras/Raf/MEK/ERK signaling cascade. Our research defined the peptide's properties and pattern of expression in HCC and identified a novel peptide, PDLC, with a function in encouraging HCC progression, offering an entirely new potential therapeutic target the disease., (© 2024. The Author(s).)

Secreted signaling peptides are central regulators of growth, development, and stress responses, but specific steps in the evolution of these peptides and their receptors are not well understood. Also, the molecular mechanisms of peptide-receptor binding are only known for a few examples, primarily owing to the limited availability of protein structural determination capabilities to few laboratories worldwide. Plants have evolved a multitude of secreted signaling peptides and corresponding transmembrane receptors. Stress-responsive SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs) were recently identified. Bioactive SCOOPs are proteolytically processed by subtilases and are perceived by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) in the model plant Arabidopsis thaliana . How SCOOPs and MIK2 have (co)evolved, and how SCOOPs bind to MIK2 are unknown. Using in silico analysis of 350 plant genomes and subsequent functional testing, we revealed the conservation of MIK2 as SCOOP receptor within the plant order Brassicales. We then leveraged AI-based structural modeling and comparative genomics to identify two conserved putative SCOOP-MIK2 binding pockets across Brassicales MIK2 homologues predicted to interact with the "SxS" motif of otherwise sequence-divergent SCOOPs. Mutagenesis of both predicted binding pockets compromised SCOOP binding to MIK2, SCOOP-induced complex formation between MIK2 and its coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1, and SCOOP-induced reactive oxygen species production, thus, confirming our in silico predictions. Collectively, in addition to revealing the elusive SCOOP-MIK2 binding mechanism, our analytic pipeline combining phylogenomics, AI-based structural predictions, and experimental biochemical and physiological validation provides a blueprint for the elucidation of peptide ligand-receptor perception mechanisms., Competing Interests: Competing interests statement:The authors declare no competing interest.

Enabling the precise control of protein functions with artificially programmed reaction patterns is beneficial for investigating biological processes. Although several strategies have been established that employ the programmability of nucleic acid, they have been limited to DNA hybridization without external stimuli or target binding. Here, we report an approach for the DNA-mediated control of the tripartite split-GFP assembly via aptamers with responsiveness to intracellular small molecules as stimuli. We designed a novel structure-switching aptamer-peptide conjugate as a hetero modulator for split GFP in response to ATP. By conjugating two peptides (S10/11) derived from the tripartite split-GFP to ATP aptamer, we achieved GFP reassembly using only ATP as a trigger molecule. The response to ATP at ≥4 mM concentrations indicated that it can be applied to respond to intracellular ATP in live cells. Furthermore, our hetero-modulator exhibited high and long-term stability, with a half-life of approximately four days in a serum stability assay, demonstrating resistance to nuclease degradation. We validated that our aptamer-modulator split GFP was successfully reconstituted in the cell in response to intracellular ATP levels. Our aptamer-modulated split GFP platform can be utilized to monitor a wide range of intracellular metabolites by replacing the aptamer sequence., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one source, 5' untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends. Almost all of these N-terminal extensions are from highly abundant genes, which suggests that the novel regions we detect are just the tip of the iceberg. These upstream regions have characteristics that are not typical of coding exons. Their GC-content is remarkably high, even higher than 5' regions in other genes, and a large majority have non-canonical start codons. Although some novel upstream regions have cross-species conservation - five have orthologues in invertebrates for example - the reading frames of two thirds are not conserved beyond simians. These non-conserved regions also have no evidence of purifying selection, which suggests that much of this translation is not functional. In addition, non-conserved upstream regions have significantly more peptides in cancer cell lines than would be expected, a strong indication that an aberrant or noisy translation initiation process may play an important role in translation from upstream regions., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B ∗ 5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8 + T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B ∗ 5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control., Competing Interests: Declaration of interests B.D.W. and G.D.G have filed patent application PCT/US2020/022403. G.D.G. also reports research funding from Moderna and Merck., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ferroptosis, a distinctive form of programmed cell death, has been implicated in numerous pathological conditions, and its inhibition is considered a promising therapeutic strategy. Currently, there is a scarcity of efficient antagonists for directly regulating intracellular ferrous iron. Ferritinophagy, an essential process for supplying intracellular labile iron, relies on nuclear receptor coactivator 4 (NCOA4), a selective autophagy receptor for the ferritin iron storage complex, thus playing a pivotal role in ferritinophagy. In this study, we reported a novel von Hippel-Lindau-based NCOA4 degrader, V3 , as a potent ferroptosis inhibitor with an intracellular ferrous iron inhibition mechanism. V3 significantly reduced NCOA4 levels and downregulated intracellular ferrous iron (Fe 2+ ) levels, thereby effectively suppressing ferroptosis induced by multiple pathways within cells and alleviating liver damage. This research presents a chemical knockdown tool targeting NCOA4 for further exploration into intracellular ferrous iron in ferroptosis, offering a promising therapeutic avenue for ferroptosis-related acute liver injury.

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development., Competing Interests: Declaration of interests V.H. owns stock of Marker Therapeutics and Allovir. B.Z. received a consulting fee from AstraZeneca., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)