Funneling modulatory peptide design with generative models: Discovery and characterization of disruptors of calcineurin protein-protein interactions.

Design of peptide binders is an attractive strategy for targeting "undruggable" protein-protein interfaces. Current design protocols rely on the extraction of an initial sequence from one known protein interactor of the target protein, followed by in-silico or in-vitro mutagenesis-based optimization of its binding affinity. Wet lab protocols can explore only a minor portion of the vast sequence space and cannot efficiently screen for other desirable properties such as high specificity and low toxicity, while in-silico design requires intensive computational resources and often relies on simplified binding models. Yet, for a multivalent protein target, dozens to hundreds of natural protein partners already exist in the cellular environment. Here, we describe a peptide design protocol that harnesses this diversity via a machine learning generative model. After identifying putative natural binding fragments by literature and homology search, a compositional Restricted Boltzmann Machine is trained and sampled to yield hundreds of diverse candidate peptides. The latter are further filtered via flexible molecular docking and an in-vitro microchip-based binding assay. We validate and test our protocol on calcineurin, a calcium-dependent protein phosphatase involved in various cellular pathways in health and disease. In a single screening round, we identified multiple 16-length peptides with up to six mutations from their closest natural sequence that successfully interfere with the binding of calcineurin to its substrates. In summary, integrating protein interaction and sequence databases, generative modeling, molecular docking and interaction assays enables the discovery of novel protein-protein interaction modulators.

Antifungal recombinant psoriasin of human origin effectively inhibits fungal growth on denture base.

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.

Nuclear P38: Roles in Physiological and Pathological Processes and Regulation of Nuclear Translocation.

The p38 mitogen-activated protein kinase (p38MAPK, termed here p38) cascade is a central signaling pathway that transmits stress and other signals to various intracellular targets in the cytoplasm and nucleus. More than 150 substrates of p38α/ß have been identified, and this number is likely to increase. The phosphorylation of these substrates initiates or regulates a large number of cellular processes including transcription, translation, RNA processing and cell cycle progression, as well as degradation and the nuclear translocation of various proteins. Being such a central signaling cascade, its dysregulation is associated with many pathologies, particularly inflammation and cancer. One of the hallmarks of p38α/ß signaling is its stimulated nuclear translocation, which occurs shortly after extracellular stimulation. Although p38α/ß do not contain nuclear localization or nuclear export signals, they rapidly and robustly translocate to the nucleus, and they are exported back to the cytoplasm within minutes to hours. Here, we describe the physiological and pathological roles of p38α/ß phosphorylation, concentrating mainly on the ill-reviewed regulation of p38α/ß substrate degradation and nuclear translocation. In addition, we provide information on the p38α/ß 's substrates, concentrating mainly on the nuclear targets and their role in p38α/b functions. Finally, we also provide information on the mechanisms of nuclear p38α/b translocation and its use as a therapeutic target for p38α/ß-dependent diseases.

Nature-inspired peptide of MtDef4 C-terminus tail enables protein delivery in mammalian cells.

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.

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium.

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 Escherichiacoli 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.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.