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Protein poly‐ and oligophosphorylation are recently discovered post‐translational modifications that remain poorly characterized due to (1) the difficulty of extracting endogenously polyphosphorylated species without degradation and (2) the absence of synthetic and analytical tools to prepare and characterize poly‐ and oligophosphorylated species in biochemical contexts. Herein, we report a methodology for the selective oligophosphorylation of peptides with monodisperse phosphate chain lengths (Pn=3–6). A library of oligophosphorimidazolide (oligoP‐imidazolide) reagents featuring benzyl and o‐nitrophenylethyl protecting groups was synthesized in moderate‐to‐good yields (65–93 %). These oligoP‐imidazolide reagents enabled the selective and simultaneous conjugation of multiple phosphate units to phosphoryl nucleophiles, circumventing tedious iterative processes. The generalizability of this approach is illustrated by a substrate scope study that includes several biologically relevant phosphopeptide sequences, culminating in the synthesis of >60 examples of peptide oligophosphates (Pn=2–6). Moreover, we report the preparation of oligoP‐diimidazolides (Pn=3–5) and discuss their application in generating unique condensed phosphate‐peptide conjugates. We also demonstrate that human phospho‐ubiquitin (pS65‐Ub) is amenable to functionalization by our reagents. Overall, we envision the methods described here will enable future studies that characterize these newly discovered but poorly understood phosphorylation modes. [ABSTRACT FROM AUTHOR]

The development of therapies followed a generalized approach for a long time, assuming that a single treatment could effectively address various patient populations. However, recent breakthroughs have revealed the limitations of this one-size-fits-all paradigm. More recently, the field of therapeutics has witnessed a shift toward other modules, including cell therapies, high molecular weight remedies, personalized medicines, and gene therapies. Such advancements in therapeutic modules have the potential to revolutionize healthcare and pave the way for medicines that are more efficient and with minimal side effects. Cell therapies have gained considerable attention in regenerative medicine. Stem cell-based therapies, for instance, hold promise for tissue repair and regeneration, with ongoing research focusing on enhancing their efficacy and safety. High molecular weight drugs like peptides and proteins emerged as promising therapeutics because of their high specificity and diverse biological functions. Engineered peptides and proteins are developed for targeted drug delivery, immunotherapy, and disease-modulation. In personalized medicine, tailored treatments to individuals based on specific genetic profiling, lifestyle, biomarkers, and disease characteristics are all implemented. Clinicians have tailored treatments to optimize outcomes and minimize adverse effects, using targeted therapies based on specific mutations, yielding remarkable results. Gene therapies have revolutionized the treatment of genetic disorders by directly targeting the underlying genetic abnormalities. Innovative techniques, such as CRISPR-Cas9 have allowed precise gene editing, opening up possibilities for curing previously incurable conditions. In conclusion, advancements in therapeutic modules have the potential to revolutionize healthcare and pave the way for medicines that are more efficient and with minimal side effects. [ABSTRACT FROM AUTHOR]

Optical microcavities, particularly whispering gallery mode (WGM) microcavities enhanced by plasmonic nanorods, are emerging as powerful platforms for single‐molecule sensing. However, the impact of optical forces from the plasmonic near field on analyte molecules is inadequately understood. Using a standard optoplasmonic WGM single‐molecule sensor to monitor two enzymes, both of which undergo an open‐to‐closed‐to‐open conformational transition, the work done on an enzyme by the WGM sensor as atoms of the enzyme move through the electric field gradient of the plasmonic hotspot during conformational change has been quantified. As the work done by the sensor on analyte enzymes can be modulated by varying WGM intensity, the WGM microcavity system can be used to apply free energy penalties to regulate enzyme activity at the single‐molecule level. The findings advance the understanding of optical forces in WGM single‐molecule sensing, potentially leading to the capability to precisely manipulate enzyme activity at the single‐molecule level through tailored optical modulation. [ABSTRACT FROM AUTHOR]

Implantable drug delivery systems formed upon injection offer a host of advantages, including localized drug administration, sustained release, minimized side effects, and enhanced patient compliance. Among the various techniques utilized for the development of in situ forming drug implants, solvent-induced phase inversion emerges as a particularly promising approach. However, synthetic polymer-based implants have been associated with undesirable effects arising from polymer degradation. In response to this challenge, a novel category of drug delivery systems, known as phospholipids-based phase separation gels (PPSGs), has emerged. These gels, characterized by their low initial viscosity, exhibit injectability and undergo rapid transformation into in situ implants when exposed to an aqueous environment. A typical PPSG formulation comprises biodegradable components, such as phospholipids, pharmaceutical oil, and a minimal amount of ethanol. The minimized organic solvents in the composition show good biocompatibility. And the relatively simple composition holds promise for industrial-scale manufacturing. This comprehensive review provides an overview of the principles and advancements in PPSG systems, with specific emphasis on their suitability as drug delivery systems for a wide range of active pharmaceutical ingredients (APIs), spanning from small molecules to peptides and proteins. Additionally, we explore the critical parameters and underlying principles governing the formulation of PPSG-based drug delivery strategies, offering valuable insights on optimization strategies. [ABSTRACT FROM AUTHOR]

Biosorption mediated by peptides or proteins has emerged as an efficient and sustainable alternative for selective rare earth element (REE) recovery from waste streams, and will diversify REE supply while reducing the environmental burden. REE-binding peptides/proteins can be created and identified by using the state-of-the-art approaches, including phage display, rational design, proteomics, and homology modeling, and can be optimized by protein engineering for improved functionality and desirable properties. The activity of REE-binding peptides and proteins can be harnessed by engineering robust and reusable biomaterials for the selective recovery of REEs. The escalating global demand for rare earth elements (REEs) and the overabundance of REE-containing waste require innovative technologies for REE recovery from waste to achieve a sustainable supply of REEs while reducing the environmental burden. Biosorption mediated by peptides or proteins has emerged as a promising approach for selective REE recovery. To date, multiple peptides and proteins with high REE-binding affinity and selectivity have been discovered, and various strategies are being exploited to engineer robust and reusable biosorptive materials for selective REE recovery. This review highlights recent advances in discovering and engineering peptides and proteins for REE recovery. Future research prospects and challenges are also discussed. [ABSTRACT FROM AUTHOR]

Abstract Optical microcavities, particularly whispering gallery mode (WGM) microcavities enhanced by plasmonic nanorods, are emerging as powerful platforms for single‐molecule sensing. However, the impact of optical forces from the plasmonic near field on analyte molecules is inadequately understood. Using a standard optoplasmonic WGM single‐molecule sensor to monitor two enzymes, both of which undergo an open‐to‐closed‐to‐open conformational transition, the work done on an enzyme by the WGM sensor as atoms of the enzyme move through the electric field gradient of the plasmonic hotspot during conformational change has been quantified. As the work done by the sensor on analyte enzymes can be modulated by varying WGM intensity, the WGM microcavity system can be used to apply free energy penalties to regulate enzyme activity at the single‐molecule level. The findings advance the understanding of optical forces in WGM single‐molecule sensing, potentially leading to the capability to precisely manipulate enzyme activity at the single‐molecule level through tailored optical modulation.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH−)]N(TRIL2WL23H)3n+ that behaves similarly to native Co(II) substituted human‐CAs. Like the Zn(II) analogue, the cobalt‐derivative at slightly basic pH is incapable of hydrolyzing p‐nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH−)]N(TRIL2WL23H)3+ peptide complex. X‐ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5‐coordinate species by pH 11. UV‐vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5‐coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process. [ABSTRACT FROM AUTHOR]

Implantable drug delivery systems formed upon injection offer a host of advantages, including localized drug administration, sustained release, minimized side effects, and enhanced patient compliance. Among the various techniques utilized for the development of in situ forming drug implants, solvent-induced phase inversion emerges as a particularly promising approach. However, synthetic polymer-based implants have been associated with undesirable effects arising from polymer degradation. In response to this challenge, a novel category of drug delivery systems, known as phospholipids-based phase separation gels (PPSGs), has emerged. These gels, characterized by their low initial viscosity, exhibit injectability and undergo rapid transformation into in situ implants when exposed to an aqueous environment. A typical PPSG formulation comprises biodegradable components, such as phospholipids, pharmaceutical oil, and a minimal amount of ethanol. The minimized organic solvents in the composition show good biocompatibility. And the relatively simple composition holds promise for industrial-scale manufacturing. This comprehensive review provides an overview of the principles and advancements in PPSG systems, with specific emphasis on their suitability as drug delivery systems for a wide range of active pharmaceutical ingredients (APIs), spanning from small molecules to peptides and proteins. Additionally, we explore the critical parameters and underlying principles governing the formulation of PPSG-based drug delivery strategies, offering valuable insights on optimization strategies.

Nigella sativa, a medicinal plant with a long history of traditional use, is renowned for its wide range of pharmacological properties. Peptides and proteins extracted from Nigella sativa have shown to possess various bioactive potentials including antimicrobial activity, anticancer and anti-inflammatory, properties. This study aims to explore the bioactive potential, antimicrobial properties, and toxicity of peptides and proteins extracted from Nigella sativa for drug discovery. The peptides and proteins are extracted using various techniques including solvent extraction, enzymatic hydrolysis, and size exclusion chromatography. The extracted peptides and proteins are screened for their bioactive potential using in vitro assays such as cell viability assays, enzyme inhibition assays, and cytokine release assays. The antimicrobial properties of the peptides and proteins are evaluated using minimum inhibitory concentration (MIC) assays against various bacteria and fungi. The toxicity of the peptides and proteins is assessed using in vitro and in vivo assays. The results potentially contribute to the development of novel drugs from Nigella sativa peptides and proteins. [ABSTRACT FROM AUTHOR]

Oral administration of therapeutic peptides is limited by poor intestinal absorption. Use of engineered microorganisms as drug delivery vehicles can overcome the challenges faced by conventional delivery methods. The potential of engineered microorganisms to act synergistically with the therapeutics they deliver opens new horizons for noninvasive treatment modalities. This study engineered a probiotic yeast, Saccharomyces boulardii, to produce cell-penetrating peptides (CPPs) in situ for enhanced intestinal permeability. Four CPPs were integrated into the yeast chromosome: RRL helix, Shuffle, Penetramax, and PN159. In vitro tests on a Caco-2 cell model showed that three CPP-producing strains increased permeability without causing permanent damage. In vivo experiments on mice revealed that Sb PN159 administration over 10 days significantly increased FITC-dextran translocation into the bloodstream without causing inflammation. This study demonstrates, for the first time, the ability of an engineered microorganism to modulate host permeability for improved intestinal absorption of a macromolecule., Competing Interests: Declaration of interests H.G, K.A.H, R.V-U, and M.O.A.S have filed patents related to this work. The remaining authors have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Owing to the role of the 20S proteasome in a wide spectrum of pathologies, including neurodegenerative disorders, proteasome-associated autoinflammatory syndromes (PRAAS), and cardiovascular diseases, understanding how its structure and composition contribute to dysfunction is crucial. As a 735 kDa protein assembly, the 20S proteasome facilitates normal cellular proteostasis by degrading oxidized and misfolded proteins. Declined proteasomal activity, which can be attributed to perturbations in the structural integrity of the 20S proteasome, is considered one of the main contributors to multiple proteasome-related diseases. Devising methods to characterize the structures of 20S proteasomes provides necessary insight for the development of drugs and inhibitors that restore proper proteasomal function. Here, native mass spectrometry was combined with multiple dissociation techniques, including ultraviolet photodissociation (UVPD), to identify the protein subunits comprising the 20S proteasome. UVPD, demonstrating an ability to uncover structural features of large (>300 kDa) macromolecular complexes, provided complementary information to conventional collision-based methods. Additionally, variable-temperature electrospray ionization was combined with UV photoactivation to study the influence of solution temperature on the stability of the 20S proteasome.