Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

A Versatile Method for Site-Specific Chemical Installation of Aromatic Posttranslational Modification Analogs into Proteins.

Posttranslational modifications (PTMs) of proteins play central roles in regulating the protein structure, interactome, and functions. A notable modification site is the aromatic side chain of Tyr, which undergoes modifications such as phosphorylation and nitration. Despite the biological and physiological importance of Tyr-PTMs, our current understanding of the mechanisms by which these modifications contribute to human health and disease remains incomplete. This knowledge gap arises from the absence of natural amino acids that can mimic these PTMs and the lack of synthetic tools for the site-specific introduction of aromatic PTMs into proteins. Herein, we describe a facile method for the site-specific chemical installation of aromatic PTMs into proteins through palladium-mediated S-C(sp2) bond formation under ambient conditions. We demonstrate the incorporation of novel PTMs such as Tyr-nitration and phosphorylation analogs to synthetic and recombinantly expressed Cys-containing peptides and proteins within minutes and in good yields. To demonstrate the versatility of our approach, we employed it to prepare 10 site-specifically modified proteins, including nitrated and phosphorylated analogs of Myc and Max proteins. Furthermore, we prepared a focused library of site-specifically nitrated and phosphorylated α-synuclein (α-Syn) protein, which enabled, for the first time, deciphering the role of these competing modifications in regulating α-Syn conformation aggregation in vitro. Our strategy offers advantages over synthetic or semisynthetic approaches, as it enables rapid and selective transfer of rarely explored aromatic PTMs into recombinant proteins, thus facilitating the generation of novel libraries of homogeneous posttranslationally modified proteins for biomarker discovery, mechanistic studies, and drug discovery.

Exocyclic and Linker Editing of Lys63-linked Ubiquitin Chains Modulators Specifically Inhibits Non-homologous End-joining Repair.

Despite the great advances in discovering cyclic peptides against protein targets, their reduced aqueous solubility, cell permeability, and activity of the cyclic peptide restrict its utilization in advanced biological research and therapeutic applications. Here we report on a novel approach of structural alternation of the exocyclic and linker parts that led to a new derivative with significantly improved cell activity allowing us to dissect its mode of action in detail. We have identified an effective cyclic peptide (CP7) that induces approximately a 9-fold increase in DNA damage accumulation and a remarkable increase in apoptotic cancer cell death compared to the reported molecule. Notably, treating cells with CP7 leads to a dramatic decrease in the efficiency of non-homologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs), which is accompanied by an increase in homologous recombination (HR) repair. Interestingly, treating BRCA1-deficient cells with CP7 restores HR integrity, which is accompanied by increased resistance to CP7. Additionally, CP7 treatment increases the sensitivity of cancer cells to ionizing radiation. Collectively, our findings demonstrate that CP7 is a selective inhibitor of NHEJ, offering a potential strategy to enhance the effectiveness of radiation therapy.

Review on Uric Acid Recognition by MOFs with a Future in Machine Learning.

Uric acid (UA) is produced from purine metabolism and serves as a prevalent biomarker for multiple diseases including cancer. Hyperuricemia or hypouricemia can cause multiple dysfunctions throughout the biological processes. Consequently, there is a pressing need for monitoring UA concentration in body fluid. While clinical methods are known, the availability of a point-of-care testing (PoCT) kit remains conspicuously absent. In the case of electrochemical recognition of UA, the oxidation potential of ascorbic acid closely aligns with that of UA and thus it hinders the detection process, which eventually may result in false positive signals. Several chemosensors are known in the field of supramolecular chemistry, and metal-organic frameworks (MOFs) are one of the best-performing contenders due to their robustness, stability, and versatile structures. In this review, we tried to unbox the up-to-date development of UA sensing by MOFs. We delve into the state of UA recognition by MOFs, exploring both electrochemical and fluorometric pathways and drawing comparisons with structurally similar probes like covalent organic frameworks (COFs) to understand/establish the advantages of MOFs specifically in UA sensing. In the absence of a PoCT kit, we have provided the conceptual outlook for designing a PoCT device termed a "Urimeter" via electrochemical operation. For the first time, we have proposed different methods of how UA sensing can be tied up with artificial intelligence and machine learning (AI-ML).

Structure-Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell-Penetrating Peptides for Live-Cell Delivery of Synthetic Proteins.

Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

Probing the cell delivery of synthetic diubiquitin chains.

In this study, the live-cell delivery of structurally different synthetic diubiquitin chains was examined. We found that the combination of structural variations of the Ub chains (intrinsic factors); nature of CPP and CPP-protein linkage (extrinsic factors) influence their delivery.

Gold(I)-Mediated Rapid Cyclization of Propargylated Peptides via Imine Formation.

In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30-60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov's addition of the amine on the Au(I)-activated propargyl. This strategy was successfully applied to prepare efficiently (56-94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.

Highly Efficient Cyclization Approach of Propargylated Peptides via Gold(I)-Mediated Sequential C-N, C-O, and C-C Bond Formation.

A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains.

Synthesis and delivery of a stable phosphorylated ubiquitin probe to study ubiquitin conjugation in mitophagy.

Protein post-translational modifications are involved in essentially all aspects of cellular signaling. Their dynamic nature and the difficulties in installing them using enzymatic approaches limits their direct study in human cells. Reported herein is the first synthesis, delivery and cellular study of a stable phosphoubiquitin probe. Our results compare Parkin's substrate preference during mitophagy via direct visualization of a phosphorylated ubiquitin probe in the cellular environment.

Enhanced Live-Cell Delivery of Synthetic Proteins Assisted by Cell-Penetrating Peptides Fused to DABCYL.

Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.