Formation Of Covalent Complex Research Articles

Hemolymph protease-17b activates proHP6 to stimulate melanization and Toll signaling in Manduca sexta

Manduca sexta hemolymph protease-6 (HP6) plays a central role in coordinating antimicrobial responses, such as prophenoloxidase (PPO) activation and Toll signaling. Our previous studies indicated that HP5 and GP6 activate proHP6 in larval hemolymph and extraembryonic tissues, respectively. Here, we report the characterization of HP17b as another HP6 activating enzyme and its regulation by multiple serpins in hemolymph. The precursor of HP17b expressed in baculovirus infected Sf9 cells became spontaneously cleaved at two sites, and these products were purified together in one preparation named HP17b′, a mixture of proHP17b, a 35 kDa intermediate, and HP17b. HP17b′ converted proHP6 to HP6. As reported before, HP6 converted precursors of PPO activating protease-1 (PAP1) and HP8 to their active forms. HP8 activates proSpӓtzle-1 to turn on Toll signaling. We found HP17b′ directly activated proSPHI and II to form a cofactor for PPO activation by PAP1. Supplementation of larval hemolymph with HP17b′, HP17b, or proHP17b significantly increased PPO activation. Adding Micrococcus luteus to the reactions did not enhance PPO activation in the reactions containing HP17b′, HP17b, or proHP17b. Using HP17b antibodies, we isolated from induced plasma HP17b fragments and associated proteins (e.g., serpin-4). Serpin-1A, 1J, 1J′, 4, 5, or 6 reduced the activation of proHP6 by HP17b’ through formation of covalent complexes with active HP17b. We detected an activity for proHP17b cleavage in hemolymph from bar-stage pharate pupae but failed to purify the protease due to its high instability. Other known HPs did not activate proHP17b in vitro. Together, these results suggest that HP17b is a clip-domain protease activated by an unknown endopeptidase in response to a danger signal and regulated by multiple serpins.

Design Optimization of a Novel Catalytic Approach for Transglucosylated Isomaltooligosaccharides into Dietary Polyols Structures by Leuconostoc mesenteroides Dextransucrase.

Polyols, or sugar alcohols, are widely used in the industry as sweeteners and food formulation ingredients, aiming to combat the incidence of diet-related Non-Communicable Diseases. Given the attractive use of Generally Regarded As Safe (GRAS) enzymes in both academia and industry, this study reports on an optimized process to achieve polyols transglucosylation using a dextransucrase enzyme derived from Leuconostoc mesenteroides. These enzyme modifications could lead to the creation of a new generation of glucosylated polyols with isomalto-oligosaccharides (IMOS) structures, potentially offering added functionalities such as prebiotic effects. These reactions were guided by a design of experiment framework, aimed at maximizing the yields of potential new sweeteners. Under the optimized conditions, dextransucrase first cleared the glycosidic bond of sucrose, releasing fructose with the formation of an enzyme-glucosyl covalent intermediate complex. Then, the acceptor substrate (i.e., polyols) is bound to the enzyme-glucosyl intermediate, resulting in the transfer of glucosyl unit to the tested polyols. Structural insights into the reaction products were obtained through nuclear maneic resonance (NMR) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses, which revealed the presence of linear α(1 → 6) glycosidic linkages attached to the polyols, yielding oligosaccharide structures containing from 4 to 10 glucose residues. These new polyols-based oligosaccharides hold promise as innovative prebiotic sweeteners, potentially offering valuable health benefits.

Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes.

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C.

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

Cytosine analogues as DNA methyltransferase substrates.

DNA methyltransferases are drug targets for myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), acute myelogenous leukemia (AML)and possibly β-hemoglobinopathies. We characterize the interaction of nucleoside analogues in DNA with a prokaryotic CpG-specific DNA methyltransferase (M.MpeI) as a model for mammalian DNMT1 methyltransferases. We tested DNA containing 5-hydroxymethylcytosine (5hmC), 5-hydroxycytosine (5OHC), 5-methyl-2-pyrimidinone (in the ribosylated form known as 5-methylzebularine, 5mZ), 5,6-dihydro-5-azacytosine (dhaC), 5-fluorocytosine (5FC), 5-chlorocytosine (5ClC), 5-bromocytosine (5BrC)and 5-iodocytosine (5IC). Covalent complex formation was by far most efficient for 5FC. Non-covalent complexes were most abundant for dhaC and 5mZ. Surprisingly, we observed methylation of 5IC and 5BrC, and to a lesser extent 5ClC and 5FC, in the presence, but not the absence of small molecule thiol nucleophiles. For 5IC and 5BrC, we demonstrated by mass spectrometry that the reactions were due to methyltransferase driven dehalogenation, followed by methylation. Crystal structures of M.MpeI-DNA complexes capture the 'in' conformation of the active site loop for analogues with small or rotatable (5mZ) 5-substituents and its 'out' form for bulky 5-substituents. Since very similar 'in' and 'out' loop conformations were also observed for DNMT1, it is likely that our conclusions generalize to other DNA methyltransferases.

Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

Targeting yeast topoisomerase II by imidazo and triazoloacridinone derivatives resulting in their antifungal activity

Fungal pathogens are considered as serious factors for deadly diseases and are a case of medical concern. Invasive fungal infections also complicate the clinical course of COVID-19, leading to a significant increase in mortality. Furthermore, fungal strains' multidrug resistance has increased the demand for antifungals with a different mechanism of action. The present study aimed to identify antifungal compounds targeting yeast topoisomerase II (yTOPOII) derived from well-known human topoisomerase II (hTOPOII) poisons C-1305 and C-1311. Two sets of derivatives: triazoloacridinones (IKE1-8) and imidazoacridinones (IKE9-14) were synthetized and evaluated with a specific emphasis on the molecular mechanism of action. Our results indicated that their effectiveness as enzyme inhibitors was not solely due to intercalation ability but also as a result of influence on catalytic activity by the formation of covalent complexes between plasmid DNA and yTOPOII. Lysine conjunction increased the strength of the compound's interaction with DNA and improved penetration into the fungal cells. Triazoloacridinone derivatives in contrast to starting compound C-1305 exhibited moderate antifungal activity and at least twice lower cytotoxicity. Importantly, compounds (IKE5-8) were not substrates for multidrug ABC transporters whereas a derivative conjugated with lysine (IKE7), showed the ability to overcome C. glabrata fluconazole-resistance (MIC 32–64 µg mL−1).

Effect of phlorotannins modification on the physicochemical, structural and functional properties of soybean protein isolate and controlled hydrolysates: Covalent and non-covalent interactions

In this research, the physicochemical, structural and functional properties of soybean protein isolate (SPI) as well as controlled hydrolysates (HSPI) were investigated by treatment with different concentrations of phlorotannins (PT) (0, 0.2, 0.5, 1.0, 2.0, mg/mL) through covalent and non-covalent interactions. Native/SDS-PAGE confirmed the formation of covalent and non-covalent complexes between PT and SPI/HSPI, and the presence of covalent bonds other than disulfide bonds in the covalent complexes (C–N, C–S bonds). After binding to PT, the secondary and tertiary structures of SPI/HSPI were changed. The secondary structure of complexes was transformed from β-sheet and β-turn to α-helix and random coil, and higher rates of transformation for covalent complexes. With the increase of PT concentration, the fluorescence and UV intensity of covalent/non-covalent complexes gradually reduced and rose, respectively; covalent complexes had lower fluorescence and higher UV intensity at equal concentrations. The particle size and ζ-potential of covalent/non-covalent complexes rose with PT concentration, and the PDI decreased and then rose, reaching a minimum value of 0.21 (HSPI-PT covalent) at PT of 0.5 mg/mL. TEM showed PT addition induced the aggregation of the complexes to increase. Meanwhile, surface hydrophobicity reduced while the antioxidant activity rose. Solubility, foaming activity and emulsifying activity of HSPI-PT covalent complexes first rose and then declined, and were optimal at PT of 0.5 mg/mL, with 78%, 250% and 97 m2/g, respectively. These will provide references for the improvement of soy protein properties and the application of complexes in formulated foods.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.

Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS-TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate-TE interaction during the macrocyclization step are still unknown. To understand the TE-mediated macrocyclization, here we report the development of a substrate-based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p-nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)-TE containing tyrocidine synthetase.

Influence of environmental pH on the interaction properties of WP-EGCG non-covalent nanocomplexes.

Whey protein-epigallocatechin gallate (WP-EGCG) covalent conjugates and non-covalent nanocomplexes were prepared and compared using Fourier-transform infrared spectra. The effect of pH (at 2.6, 6.2, 7.1, and 8.2) on the non-covalent nanocomplexes' functional properties and the WP-EGCG interactions were investigated by studying antioxidant activity, emulsification, fluorescence quenching, and molecular docking, respectively. With the formation of non-covalent and covalent complexes, the amide band decreased; the -OH peak disappeared; the antioxidant activity of WP-EGCG non-covalent complexes was 2.59- and 2.61-times stronger than WP-EGCG covalent conjugates for 1-diphenyl-2-picryl-hydrazyl (DPPH) and ferric reducing ability of plasma(FRAP), respectively (particle size: 137 versus 370 nm). The antioxidant activity (DPPH 27.48-44.32%, FRAP 0.47-0.63) was stronger at pH 6.2-7.1 than at pH 2.6 and pH 8.2 (DPPH 19.50% and 26.36%, FRAP 0.39 and 0.41). Emulsification was highest (emulsifying activity index 181 m2 g-1 , emulsifying stability index 107%) at pH 7.1. The interaction between whey protein (WP) and EGCG was stronger under neutral and weakly acidic conditions: KSV (5.11-8.95 × 102 L mol-1 ) and Kq (5.11-8.95 × 1010 L mol s-1 ) at pH 6.2-7.1. Binding constants (pH 6.2 and pH 7.1) increased with increasing temperature. Molecular docking suggested that hydrophobic interactions played key roles at pH 6.2 and pH 7.1 (∆H > 0, ∆S > 0). Hydrogen bonding was the dominant force at pH 2.6 and pH 8.2 (∆H < 0, ∆S < 0). Environmental pH impacted the binding forces of WP-EGCG nanocomplexes. The interaction between WP and EGCG was stronger under neutral and weakly acidic conditions. Neutral and weakly acidic conditions are preferable for WP-EGCG non-covalent nanocomplex formation. © 2023 Society of Chemical Industry.

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein-Protein Interaction as a Molecular Glue Drug Discovery Approach.

Molecules that stabilize protein-protein interactions (PPIs) are invaluable as tool compounds for biophysics and (structural) biology, and as starting points for molecular glue drug discovery. However, identifying initial starting points for PPI stabilizing matter is highly challenging, and chemical optimization is labor-intensive. Inspired by chemical crosslinking and reversible covalent fragment-based drug discovery, we developed an approach that we term "molecular locks" to rapidly access molecular glue-like tool compounds. These dual-covalent small molecules reversibly react with a nucleophilic amino acid on each of the partner proteins to dynamically crosslink the protein complex. The PPI between the hub protein 14-3-3 and estrogen-related receptor γ (ERRγ) was used as a pharmacologically relevant case study. Based on a focused library of dual-reactive small molecules, a molecular glue tool compound was rapidly developed. Biochemical assays and X-ray crystallographic studies validated the ternary covalent complex formation and overall PPI stabilization via dynamic covalent crosslinking. The molecular lock approach is highly selective for the specific 14-3-3/ERRγ complex, over other 14-3-3 complexes. This selectivity is driven by the interplay of molecular reactivity and molecular recognition of the composite PPI binding interface. The long lifetime of the dual-covalent locks enabled the selective stabilization of the 14-3-3/ERRγ complex even in the presence of several other competing 14-3-3 clients with higher intrinsic binding affinities. The molecular lock approach enables systematic, selective, and potent stabilization of protein complexes to support molecular glue drug discovery.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

N-terminal domain of tyrosyl-DNA phosphodiesterase I regulates topoisomerase I-induced toxicity in cells

Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes phosphodiester-linked adducts from both ends of DNA. This includes the topoisomerase I (TOP1)-DNA covalent reaction intermediate that is the target of the camptothecin class of chemotherapeutics. Tdp1 two-step catalysis is centered on the formation of a Tdp1-DNA covalent complex (Tdp1cc) using two catalytic histidines. Here, we examined the role of the understudied, structurally undefined, and poorly conserved N-terminal domain (NTD) of Tdp1 in context of full-length protein in its ability to remove TOP1cc in cells. Using toxic Tdp1 mutants, we observed that the NTD is critical for Tdp1’s ability to remove TOP1-DNA adducts in yeast. Full-length and N-terminal truncated Tdp1 mutants showed similar expression levels and cellular distribution yet an inversed TOP1-dependent toxicity. Single turnover catalysis was significantly different between full-length and truncated catalytic mutants but not wild-type enzyme, suggesting that Tdp1 mutants depend on the NTD for catalysis. These observations suggest that the NTD plays a critical role in the regulation of Tdp1 activity and interaction with protein-DNA adducts such as TOP1cc in cells. We propose that the NTD is a regulatory domain and coordinates stabilization of the DNA-adducted end within the catalytic pocket to access the phosphodiester linkage for hydrolysis.

Sonochemical effects on fabrication, characterization and antioxidant activities of β-lactoglobulin-chlorogenic acid conjugates.

The β-lactoglobulin-chlorogenic acid (LG-CA) conjugate was explored to be formed through ultrasonication, redox-pair method and their combination, the ultrasonication used a probe ultrasonic machine with a 6mm probe at 270W, and the frequency was 20-25kHz. The formation of the conjugate was confirmed by SDS-PAGE with a larger molecular weight. Besides, Fourier infrared spectroscopy (FTIR) and Circular dichroism (CD) indicated changes in the secondary structure of the LG-CA conjugate. The α-helix and β-sheet contents of LG decreased and the unordered content increased significantly after the formation of covalent complexes. In addition, both the ultrasonic treatment and its combination with redox-pair method could significantly improve the antioxidant properties of LG. The former increased to 23.16μmol Trolox/g sample, the latter 82-106μmol Trolox/g sample. Therefore, ultrasonication could be used both individually and in combination with the redox-pair method to produce LG-CA conjugates with stronger antioxidant activities.

Hierarchical subcomponent self-assembly of covalent triple-stranded complexes with 3d-4f vertices: luminescence and magnetic properties.

Well-defined 3d-4f heterometallic supramolecular architectures have attracted attention because of their applications in the field of luminescence and magnetism. However, covalent metallo-supramolecular discrete complexes, decorated with hetero-metallic vertices, have never been reported because of the difficulties in design and control. Herein, we report a series of covalent metallo-supramolecular discrete complexes with 3d-4f vertices synthesized by hierarchical subcomponent self-assembly of tris(2-aminoethyl)amine, 2,6-diformyl-p-cresol, and lanthanide ions (Ln) with different amines and transition metal ions. The programmable self-assembly process results in the formation of triple-stranded hetero-metallic covalent organic discrete complexes, namely 3a-3c-(Ln, Zn) (Ln = SmIII, EuIII, DyIII, YbIII and LuIII) and 3a'-(Dy, Co), which are characterized by nuclear magnetic resonance (NMR) analysis, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and single-crystal X-ray analysis. Photophysical investigations disclose that the organic skeleton of 3a-(Ln, Zn) exhibits an excellent sensitizing ability toward SmIII, EuIII, and YbIII ions, displaying characteristic luminescence emission in both the visible and near-infrared (NIR) regions. AC susceptibility measurements of 3a'-(Dy, Co) reveal the frequency-independent performance under zero dc field, suggesting the absence of slow relaxation of magnetization. This work offers a new approach for the fabrication of discrete metallic covalent architectures with 3d-4f vertices.

Effects of catechin types found in tea polyphenols on the structural and functional properties of soybean protein isolate–catechin covalent complexes

Soy protein isolate (SPI) has useful functional properties, such as gelation, emulsification, and foaming. However, SPI is usually dense and has a unique spherical structure, which hinders its practical application. In this study, the effects of three typical catechins (epigallocatechin gallate (EGCG), epigallocatechin (EGC), and epicatechin (EC)) on the structure and functional properties of soy protein isolate (SPI) under alkali conditions were investigated. Compared with the native SPI, the fluorescence intensity of SPI–EC, SPI–EGC, and SPI–EGCG complexes decreased, while their fluorescence quenching rate and binding sites increased, indicating the formation of covalent complexes. The SPI–EGCG complex has a higher fluorescence quenching rate and more binding sites than those of SPI–EC and SPI–EGCG complexes. The emulsifying activity and stability of the SPI–EGCG complex were 1.16 m2/g and 147 min, respectively, higher than those of latter two complexes. Hence, the functional property of SPI and the stability of catechins could be improved by the covalent interaction between the two. This study provides new insights into the relationship between the type of polyphenol and the structure of SPI.

Distinction of early complement classical and lectin pathway activation via quantification of C1s/C1-INH and MASP-1/C1-INH complexes using novel ELISAs.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

Structural and functional studies of legumain–mycocypin complexes revealed a competitive, exosite-regulated mode of interaction

Under pathophysiologic conditions such as Alzheimer’s disease and cancer, the endolysosomal cysteine protease legumain was found to translocate to the cytosol, the nucleus, and the extracellular space. These noncanonical localizations demand for a tight regulation of legumain activity, which is in part conferred by protein inhibitors. While there is a significant body of knowledge on the interaction of human legumain with endogenous cystatins, only little is known on its regulation by fungal mycocypins. Mycocypins are characterized by (i) versatile, plastic surface loops allowing them to inhibit different classes of enzymes and (ii) a high resistance toward extremes of pH and temperature. These properties make mycocypins attractive starting points for biotechnological and medical applications. In this study, we show that mycocypins utilize an adaptable reactive center loop to target the active site of legumain in a substrate-like manner. The interaction was further stabilized by variable, isoform-specific exosites, converting the substrate recognition into inhibition. Additionally, we found that selected mycocypins were capable of covalent complex formation with legumain by forming a disulfide bond to the active site cysteine. Furthermore, our inhibition studies with other clan CD proteases suggested that mycocypins may serve as broad-spectrum inhibitors of clan CD proteases. Our studies uncovered the potential of mycocypins as a new scaffold for drug development, providing the basis for the design of specific legumain inhibitors.

Designing covalent sodium caseinate-quercetin complexes to improve emulsifying properties and oxidative stability

The complexation of proteins with phenolic compounds has been considered a promising route to improve the oxidative stability of oil-in-water (O/W) emulsions. In this context, physicochemical and techno-functional properties of sodium caseinate (SC) chemically modified by the interaction with quercetin (Q) under alkaline conditions were evaluated using different molar ratios of the components (SC:Q). The formation of covalent complexes was analysed by the changes in SC structure and properties.. The results showed that the surface hydrophobicity of SC gradually decreased with increasing Q concentration. Whereas an increase in phenolic content and antioxidant activity by DPPH and FRAP was observed. The techno-functionality of the complexes was evaluated by their capacity of stabilizing oil-in-water emulsions. The emulsion stabilized by complexes and non-modified SC showed an average droplet size (D4.3) between 1,632 ± 0,068 and 1,945 ± 0,431 µm. After 21d of storage, no coalescence was observed, evidencing the formation of stable emulsions. Furthermore, the highest oxidative stability of the emulsion was observed at the highest concentration of Q, while the formation of hydroperoxides and aldehydes was greater in the emulsions stabilized with non-modified SC. The formation of primary lipid oxidation compounds was approximately 3x higher in emulsions stabilized only with SC compared to those stabilized with the SC:Q complex in a 1:1 ratio. These results suggest that modification of SC with Q is a potential approach tuning both chemical and techno-functional properties of SC.