Abstract Calcineurin (Cn) is a protein phosphatase that initiates T-cell activation by dephosphorylating the transcription factor NFAT, driving its nuclear translocation and the transcription of immune-related genes. While clinical immunosuppressants like Cyclosporine A (CsA) potently inhibit Cn, they completely block its catalytic site, leading to non-specific inhibition and severe off-target toxicity. Selectively targeting the specific protein-protein interaction (PPI) between Cn and NFAT presents a safer therapeutic strategy. We previously identified the C16orf74 (C16) peptide as a high-affinity Cn-NFAT PPI inhibitor; however, its utility in cellular systems is restricted by poor membrane permeability. In this study, we evaluated cell-penetrating peptide (CPP) conjugates of C16 with an N-terminus transactivator of transcription (TAT) and polyarginine (R11) to enable efficient intracellular delivery. Structural modeling, fluorescence polarization displacement, and pull-down assays confirmed that the CPP–C16 conjugates retain the ability to compete with an NFAT-derived peptide and bind Cn. Fluorescence microscopy demonstrated efficient intracellular entry of TAT-C16 and R11-C16 in mammalian cells, and effective inhibition of NFAT nuclear translocation and attenuation of downstream NFAT-dependent transcriptional activity of the IL-2 gene in human T cells at concentrations of 10 µM or lower. Crucially, unlike CsA, the CPP-C16 peptides exhibited minimal cytotoxicity even at high concentrations of up to 50 µM, establishing a potential safe therapeutic window. These findings establish CPP-C16 conjugates as effective, cell-permeable, and non-toxic inhibitors of the Cn-NFAT signaling axis, providing the basis for the development of PPI-directed immunosuppressants.

The loss of epigenetic information has been proposed as a driver of aging and diseases, but the reversibility and causality of this process remain underexplored. Here, we analyze liver-unique methylation sites-genomic loci that show distinct methylation patterns in the liver compared to other tissues. Upon disease progression, these sites overwhelmingly regress toward the pan-tissue average. In addition, we demonstrate that this regression also occurs in a majority of these sites during normal aging. Using causal sites previously identified by Mendelian randomization analysis, we identify significant enrichment of liver-unique methylation sites in causal aging-associated loci, particularly sites that are highly methylated in healthy liver. Remarkably, repeated fasting, a metabolic intervention known to improve liver function, partially restores the DNA accessibility patterns at these sites. This restoration also occurs in isolated hepatocytes subjected to fasting-mimicking conditions, suggesting the effect is cell-autonomous rather than due to changes in tissue composition. The liver-unique methylation sites are enriched for binding sites of key metabolic transcription factors and show significant overlap with genetic variants associated with liver disease risk, suggesting a mechanistic link between epigenetic information loss and liver dysfunction. Our findings establish epigenetic information loss as both a marker and mediator of liver aging and disease, while demonstrating its potential reversibility through metabolic interventions.

Real-time non-invasive monitoring of food spoilage using a protein-based pH-sensitive FRET biosensor.

Abstract Bispecific fusion proteins represent a unique strategy for developing precision therapeutics. By linking functional domains from distinct proteins, these biomolecules can engage multiple targets, enhancing both therapeutic efficacy and safety. Unlike bispecific antibodies, low-molecular-weight fusion proteins offer distinct advantages, including reduced immunogenicity and superior tissue penetration due to their relatively compact size and structure. Such a profile is particularly valuable in managing complex inflammatory diseases, where modulating multiple pathways is required to impart an effective anti-inflammatory effect. Among the various regulators of immune signaling, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and interleukin-10 (IL-10) play imperative roles in immune suppression through their interactions with CD80/86 and IL-10R, respectively. While Fc-fused CTLA-4 is a clinically approved drug (e.g., Abatacept), the clinical development of IL-10 has been hampered by unpredictable immunostimulatory side effects. Here, we engineered a bispecific fusion protein linking the extracellular domain of CTLA-4 to IL-10. We successfully expressed the protein in E. coli as an N-terminal GST-tagged variant and refolded it from the inclusion bodies. Additionally, we achieved soluble expression of an Fc-tagged variant in mammalian CHO cells. Both origins demonstrated binding to their cognate receptors, CD80 and IL-10R. Finally, the fusion protein demonstrated a T cell-inhibitory effect by reducing Interferon-γ (IFNγ) secretion levels in an in vitro human Virus-Specific T cells (VSTs) model. This innovative protein engineering offers a promising strategy for addressing unmet clinical needs in autoimmune and inflammatory diseases.

Abscisic acid (ABA) functions as a central regulator of dehydration responses in land plants. As such, ABA signaling was pivotal in facilitating the colonization of terrestrial habitats. The conserved ABA signal transduction module consists of 2C-type protein phosphatases (PP2Cs) and their ABA-triggered inhibitors, PYRABACTIN RESISTANCE 1-like proteins (PYLs). Recent evidence indicates that ABA perception emerged from a latent signaling pathway involving a constitutively PP2C-inhibiting PYL homolog. Consequently, ancestral ABA receptors exerted high background signaling, limiting the dynamic range of ABA-dependent signaling. In angiosperms, ABA receptor families are characteristically large and diverse and include a clade-specific subgroup whose members form homodimers, thereby assuming strict ABA dependency. Here, we show that ABA receptors in mosses originate from an independent expansion, giving rise to three subfamilies. Yeast two-hybrid and in vitro PP2C-inhibition assays indicate that moss PYLs feature low basal activities. However, size-exclusion chromatography and additional lines of evidence suggest that moss PYLs are predominantly monomeric. A combination of mutational analysis with biochemical and physiological assays reveals that the reduced basal activities of moss PYLs are achieved through unique sets of amino acid variations. Finally, introducing causal variations to dimeric receptors dramatically compromises their ABA responsiveness, suggesting that the two evolutionary trajectories are mutually exclusive. Hence, mosses appear to have evolved a parallel mechanism to mitigate the ancestrally high background signal of the core ABA perception apparatus. This convergence highlights the shared imperative of expanding the amplitude of a central, highly adaptive signaling pathway.

Monitoring food spoilage is essential for enhancing food safety and reducing waste. pH changes serve as a valuable indicator of microbial activity, and real-time pH monitoring can provide an accurate and non-invasive indication of food spoilage. The pHlameleon chimera proteins were developed for pH sensing by Förster resonance energy transfer (FRET) and were extensively used in various biomedical applications. Herein, we evaluate the mVenus–Tolles as a FRET-based biosensor for detecting pH changes as a proxy for food spoilage. The protein was fused to an N-terminal vesicle-nucleating peptide (VNP) tag and recombinantly expressed and purified to homogeneity. Experimental validation demonstrated pH-responsive FRET signal in an array of buffers as well as in a complex food matrix such as chickpea paste, correlating with increasing acidity and microbial growth in food. These findings suggest that this protein-based FRET biosensor holds promise for safe integration into food or packaging for real-time freshness monitoring.

Food spoilage is a significant economic and environmental concern, and it is estimated that ∼30% of fresh food is destroyed due to food spoilage between harvest to consumer. Current food preservatives are chemicals that are associated with various health risks and often have limited effectiveness under certain conditions like pH and temperature. Consequently, there’s a growing need to develop effective, natural, and economical food preservatives. Herein, we studied the natural protein psoriasin as a potential food preservative. Psoriasin is naturally secreted in the oral cavity and has an effective and validated antimicrobial activity which makes it a potentially effective and safe protein-based food preservative. Indeed, our preliminary results show promising antimicrobial activity of the recombinant protein against food-related microbial organisms in vitro and in various food types. In addition, it is recombinantly expressed at high levels, which could set a cost-effective manufacturing process. These results set psoriasin as a safe and effective natural food preservative, addressing consumer demand for healthier food options and reducing food waste. ### Competing Interest Statement Maayan Gal and Zvi Hayouka are the scientific co-founders of Bountica Ltd.  Israeli Ministry for Science, Technology and Space, Grant# 000735 Bountica Ltd, n/a Marian Gertner Institute for Medical Nano-Systems, n/a

Fibroblast cells are a primary source of collagen, playing a vital role in maintaining the structural integrity of the oral mucosa. Given that hypoxia upregulates collagen levels in oral mucosa-derived fibroblasts (OMDFs), this study aims to investigate the effect of inhibiting prolyl-hydroxylase domain (PHD) enzymes, negative regulators of the transcription factor hypoxia-inducible factor (HIF), on collagen-I levels under normal oxygen levels. We examined three PHD inhibitors, IOX4, Enarodustat, and Daprodustat, for their effect on collagen levels in keratinized OMDFs. While IOX4 exhibited cellular cytotoxicity, Enarodustat and Daprodustat did not affect cell viability. Further treatment with Daprodustat at 12.5 or 25 μM significantly increased collagen I level more than twofold compared to non-treated cells, whereas Enarodustat had no significant effect. Additional mechanistic studies revealed that Daprodustat treatment induced the accumulation and nuclear translocation of HIF-1α and upregulated transcripts of key collagen-modifying enzymes, including P4HA1 and P4HA2, by more than twofold. This study demonstrates that direct PHD inhibition effectively enhances collagen biosynthesis in OMDFs. The mechanism involves HIF-1α-mediated upregulation of collagen-modifying enzymes. These findings highlight the potential of repurposing clinically approved PHD inhibitors as therapeutic agents for promoting the healing and regeneration of damaged gingiva and additional tissues.

The phosphatase and tensin homolog (PTEN) is a vital protein that maintains an inhibitory brake for cellular proliferation and growth. Accordingly, PTEN loss-of-function mutations are associated with a broad spectrum of human pathologies. Despite its importance, there is currently no method to directly monitor PTEN activity with cellular specificity within intact biological systems. Here we describe the development of a FRET-based biosensor using PTEN conformation as a proxy for the PTEN activity state, for two-photon fluorescence lifetime imaging microscopy. We identify a point mutation that allows the monitoring of PTEN activity with minimal interference to endogenous PTEN signaling. We demonstrate imaging of PTEN activity in cell lines, intact Caenorhabditis elegans and in the mouse brain. Finally, we develop a red-shifted sensor variant that allows us to identify cell-type-specific PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.

Aging is a major risk factor for a plethora of diseases. The information theory of aging posits that epigenetic information loss is a principal driver of the aging process. Despite this, the connection between epigenetic information loss and disease has not been thoroughly investigated. Here, we analyzed tissue-unique methylation patterns in healthy and diseased human organs, revealing that for several diseases these patterns degrade, regressing to a mean form. We interpret this as epigenetic information loss, where tissue-unique patterns erode. Information loss is not limited to diseases. Age-related erosion of unique methylation patterns was observed in some tissues and cells, while other tissues and cells diverged away from the mean. Our findings demonstrate that analyzing methylation patterns in tissue-unique sites can effectively distinguish between patients and healthy controls at least in some diseases, and underscore the role of epigenetic information loss as a common feature in various pathological conditions.Graphical abstractTissue unique methylation pattern regress toward the mean upon disease. A single methylation site, showing low methylation in the liver and high in every other tissue, becomes more methylated in diseased livers.

OBJECTIVE:To evaluate the efficacy of recombinant psoriasin as a novel treatment for oral candidiasis by eliminating Candida albicans growth on polymethyl methacrylate denture base. MATERIALS AND METHODS:Recombinant psoriasin protein was expressed and purified from E. coli, and Candida growth was monitored in vitro with varying concentrations of psoriasin. Subsequently, denture-base polymethyl methacrylate was immersed in psoriasin's solution or voriconazole, and fungal growth on the acrylic base and in the medium was examined by scanning electron microscopy and optical density, respectively. Cellular viability of HeLa and human gingival fibroblast cells treated with psoriasin was measured by methylene blue assay. RESULTS:The findings reveal an effective antifungal activity of psoriasin, completely inhibiting Candida albicans growth in RPMI at a protein concentration above 400 nM. Immersing the polymethyl methacrylate with 50 μM psoriasin completely eradicates fungal growth. Psoriasin has low cytotoxicity in HeLa cells at a concentration higher than 12 μM and no toxic effect on human gingival fibroblasts. CONCLUSIONS:This study marks psoriasin as an effective alternative to conventional antifungal treatments for denture stomatitis and a safe alternative to chemical antifungals in dental medicine and beyond.

Collagen, the most abundant protein in the extracellular matrix of mammalian cells, is extensively needed in various biotechnological and therapeutic applications, such as tissue engineering and regeneration, cosmetics, and cultivated meat. Despite the increasing demand for natural collagen from non-animal sources, it is mainly produced from animal connective tissues. Recent research has highlighted that under hypoxia, the activation of the hypoxia-inducible factor (HIF) leads to enhanced collagen type I biosynthesis. However, under normal oxygen conditions, HIF activity is downregulated by the HIF-prolyl hydroxylase (PHD) enzyme. We, therefore, hypothesized that inhibiting PHD could elevate HIF transcriptional activity and enhance collagen biosynthesis under normoxia. Our study demonstrates that inhibiting PHD using exogenous small molecules boosts HIF activity and upregulates the key enzymes, collagen prolyl 4-hydroxylases and lysyl hydroxylases, resulting in up to 29-fold increase in collagen type I in embryonic mouse fibroblast NIH/3T3 cells. These findings suggest that targeting PHD can effectively enhance collagen production in mammalian cells. Therefore, modulating key protein signaling pathways presents a promising strategy for enhancing the production of high-yield natural collagen. ### Competing Interest Statement L.A.A. M.G. and D.R. are the co-founders of Arrakis Bio Ltd., developing animal-free collagen. All other authors declare no conflicts of interest.

Cysteine is the precursor for the biosynthesis of glutathione, a key stress-protective metabolite, and methionine, which is imperative for cell growth and protein synthesis. The exact mechanism that governs the routing of cysteine toward glutathione or methionine during stresses remains unclear. Our study reveals that under oxidative stress, methionine and glutathione compete for cysteine and that the increased oxidized glutathione (GSSG) levels under stress hinder methionine biosynthesis. Moreover, we find that inhibition occurs as GSSG binds to and accelerates the degradation of cystathionine γ-synthase, a key enzyme in the methionine synthesis pathway. Consequently, this leads to a reduction in the flux toward methionine-derived metabolites and redirects cysteine utilization toward glutathione, thereby enhancing plant protection. Our study suggests a novel regulatory feedback loop involving glutathione, methionine, and cysteine, shedding light on the plant stress response and the adaptive rerouting of cysteine. These findings offer new insights into the intricate balance of growth and protection in plants and its impact on their nutritional value due to low methionine levels under stress.

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide’s ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications.

Pesticides, especially herbicides, have revolutionized agriculture by providing energy-efficient solutions for pest control that replaces labor-intensive cultivation methods. However, the widespread evolution of pesticide resistance poses a significant challenge to current agriculture. Most pesticides function by binding to specific pockets on target enzymes, enabling a single mutation to confer resistance. An alternative approach is the disruption of protein-protein interactions (PPI), thus for resistance to occur, it requires complementary mutations on both interacting partners. Despite extensive efforts, no herbicides with new modes of action have been commercialized for decades. Thus, we focused on the discovery and design of small molecule inhibitors that target the interface of the PPI complex of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT), key plant enzymes involved in the biosynthesis of the essential amino acid cysteine. Using in silico filtering techniques on a virtual library of 30 million small molecules, we identified initial hits capable of binding OASS and interfering with its interaction with a peptide derived from SAT. Subsequently, we conducted chemical optimizations to evaluate biophysical enzyme disruption, followed by cellular and in-planta activity in plants. These new compounds described herein can serve as promising starting points for further optimization as herbicides acting on a new mode of action.### Competing Interest StatementThe research was supported by funding of Projini AgChem Ltd. R.B.S, E.C, N.D and I.B are employees of Projini AgChem Ltd. J.G and M.G are consultants to Projini AgChem Ltd. Projini is the assignee of PCT patent WO 2023/06267 HERBICIDES AND USE THEREOF. Projini Ltd was funded by Migal, the Israeli innovation authority (IIA) and the trendlines-Bayer fund.

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichia coli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

BACKGROUND: The widespread evolution of pesticide resistance poses a significant challenge to current agriculture, necessitating the discovery of molecules with new modes of action. Despite extensive efforts, no major molecules with new modes of action have been commercialized for decades. Most pesticides function by binding to specific pockets on target enzymes, enabling a single target site mutation to confer resistance. An alternative approach is the disruption of protein-protein interactions (PPI), which require complementary mutations on both interacting partners for resistance to occur. Thus, our aim is the discovery and design of small-molecule inhibitors that target the interface of the PPI complex of O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT), key obligatory interacting plant enzymes involved in the biosynthesis of the amino acid cysteine. RESULTS: By employing in silico filtering techniques on a virtual library of 30 million small molecules, we identified initial hits capable of binding OASS and interfering with its interaction with a peptide derived from SAT with a half-maximal inhibitory concentration (IC50) of 34 mu m. Subsequently, we conducted molecular chemical optimizations, generating an early lead molecule (PJ4) with an IC50 value of 4 mu m. PJ4 successfully inhibited the germination of Arabidopsis thaliana seedlings and inhibited clover growth in a pre-emergence application at an effective concentration of 4.6 kg ha(-1). CONCLUSION: These new compounds described herein can serve as promising leads for further optimization as herbicides with a new mode-of-action. This technology can be used for discovering new modes of action chemicals inhibiting all pest groups. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

AbstractDuring the past 30 yr an impasse has developed in the discovery and commercialization of synthetic herbicides with new molecular targets and novel chemistries. Similarly, there has been little success with bioherbicides, both microbial and chemical. These bioherbicides are needed to combat fast-growing herbicide resistance and to fulfill the need for more environmentally and toxicologically safe herbicides. In response to this substantial and growing opportunity, numerous start-up companies are utilizing novel approaches to provide new tools for weed management. These diverse new tools broaden the scope of discovery, encompassing advanced computational, bioinformatic, and imaging platforms; plant genome–editing and targeted protein degradation technologies; and machine learning and artificial intelligence (AI)-based strategies. This review contains summaries of the presentations of 10 such companies that took part in a symposium held at the WSSA annual meeting in 2024. Four of the companies are developing microbial bioherbicides or natural product–based herbicides, and the other six are using advanced technologies, such as AI, to accelerate the discovery of herbicides with novel molecular target sites or to develop non-GMO, herbicide-resistant crops.

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein-protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.

AbstractMethionine biosynthesis relies on the sequential catalysis of multiple enzymes.Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotrophE. colistrain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering theEscherichia colimethionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY fromCorynebacterium glutamicumfailed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY ofCorynebacterium glutamicumand transformed it into a methionine-auxotrophicE. colistrain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media without external methionine supplementation. The dominant metX mutations in the evolved methionine-autotrophsEscherichia coliwere L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotrophEscherichia colivalidated their functionality. These results show that directed enzyme-evolution enables the fast engineering of new active variants within theEscherichia colimethionine direct-sulfurylation pathway, leading to efficient complementation.