Substrate Cleavage Research Articles

Nucleotide-level linkage of transcriptional elongation and polyadenylation.

Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters - from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

Regulation of VWF Cleavage Is Mediated By Coupling ADAMTS13 D- and C- Domain Binding Sites

Introduction: The metalloprotease ADAMTS13 consists of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB domains (distal domains). ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willebrand factor (VWF) to prevent ultra-large multimeric VWF-induced platelet aggregation. We and others previously described the allosteric regulation of ADAMTS13 by which the distal domains fold back and inhibit the proximal domains. VWF D4-CK domains bind the ADAMTS13 T8-CUB region to disrupt this allostery and accelerate VWF proteolysis. We recently discovered that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs (Muia, Blood, 2019). For example, c-VWF71 (C-71), a wild-type bovine VWF domain A2-derived peptide substrate, is cleaved extremely well by many vertebrate ADAMTS13. Conversely, similar substrates prepared from wild-type human h-VWF71 (H-71) and other VWF domain A2 sequences mostly tolerated cleavage by their ADAMTS13 or additional few species-specific ADAMTS13. Although unfolded VWF A2 domain engages ADAMTS13 M, D, C, and S exosites for substrate specificity and efficient cleavage, sequence differences among the various vertebrates VWF71 substrates may hold clues about specificity determinants of ADAMTS13 substrate recognition and cleavage. Methods: Multiplex FRET ADAMTS13 assays were performed using 10 novel fluorogenic substrates. Substrates P3-P10 are chimeric substrates between C-71 and H-71; species-specific amino acid residues were swapped sequentially (Fig 1A). Plasmas from various vertebrates have been characterized and reported before. Recombinant ADAMTS13 variants were transiently expressed in HEK293 cells. Assays were performed as previously described and mean results are reported. Results: The chimeric substrates (P3-P10) were designed to substitute wild-type, species-specific amino acid residues at sites known to interact with ADAMTS13 D and C domain exosites. However, amino acid substitutions also occurred at the sites that do not interact directly with any ADAMTS13 exosite. Compared to C-71 (P2), only P3 and P6 exhibited reduced rates of cleavage by all vertebrate plasma containing ADAMTS13 (Fig 1B). The P3 substrate substituted the D binding residues from human VWF71 into C-71, whereas P6 substituted the C binding residues from human VWF71 into C-71. For P3, all ADAMTS13 assayed showed ~30-100% reduction in the rate of cleavage except for armadillo and dog plasma ADAMTS13. Likewise, P6 was cleaved ~0% to 60% slowly relative to P2 by all ADAMTS13. Substituting the D, nb-1(non-binding region 1), and nb-2 sequences from human into C-71, named P8, did not further reduce rates of cleavage compared to P3. These results suggest the nb-1 and nb-2 regions of VWF71 do not participate in protease engagement, consistent with previous studies. We wondered if swapping human VWF D- and C- binding sites to C-71 as well as the bovine's to H-71 would result in ADAMTS13 cleavage rates similar to wild-type sequences. Interestingly, there was no major change in cleavage rates when D- and C- VWF binding sites were of the same species, i.e., P9 ~ P1 and P10 ~ P2, irrespective of the peptide backbone. We also sought to know if allosteric regulation of ADAMTS13 attenuates the binding of these chimeric substrates to ADAMTS13. Pigeon (potently activated by low pH) and human plasma ADAMTS13 were activated ~15.0-28.0 and ~4.8-8.0-fold, respectively when assayed with all 10 peptides. Whether ADAMTS13 was activated or not, the ratio of cleavage for individual substrates to the parental P2 remained relatively constant (~ 0.4-1.3), implying the substrate-dependent activity is independent of allosteric activation of ADAMTS13. Conclusions: The fact that D- and C- VWF binding sites from human and bovine VWF71 cannot be combined without negatively affecting the Tyr-Met bond cleavage may suggest coupling of ADAMTS13 D and C domains to regulate its activity. It is plausible to claim that ADAMTS13 VWF D- and C- binding sites and/or ADAMTS13 D and C domains may have evolved to provide a fail-safe point for allosterically activated ADAMTS13. This may prevent substrate-activated ADAMTS13, which precedes the unraveling of the VWF domain A2, from engaging and cleaving off-targets. We conclude that VWF as a substrate serves both as an ADAMTS13 activator and activity regulator. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aptamer-Small Molecule Conjugates As Potent and Reversible Surgical Anticoagulants Targeting Thrombin

Unfractionated heparin (UFH) is the first-line anticoagulant used in millions of heart surgeries annually. The high potency of UFH is attributed to its effective activation of the antithrombin-mediated anticoagulation pathway. However, the prolonged use of heparin in heart surgeries is often associated with bleeding disorders as it depletes both patient's coagulation and anticoagulation capacity. Moreover, UFH is immunogenic, which can trigger potentially life-threatening heparin-induced thrombocytopenia (HIT) when used repeatedly. Aptamers are short oligonucleotides isolated in vitro that bind to protein or small molecule targets with high affinity and specificity. Several aptamers have been created that act as anticoagulants as they can competitively block substrate binding to anticoagulation protease exosites. Compared to UFH, aptamers have advantages, including not activating antithrombin, reversibility by administration of a well-tolerated antisense oligonucleotide, and low immunogenicity. However, the anticoagulant potency of aptamers is often limited by their inability to effectively inhibit the catalytic sites of their protease targets. Here we provide a strategy to solve this potency problem by conjugating a protease active-site inhibitor to an exosite-binding aptamer via a polynucleotide linker. The resulting bivalent conjugate binds to both the active site and exosite of the target protease to completely inhibit protease function. Meanwhile, the synergy between two binding elements significantly improves the binding affinity compared to the small molecule inhibitor or the aptamer alone. As a proof of concept, we conjugated the thrombin active site inhibitor DAB to the 5’ terminus of anti-thrombin aptamer HD22 via a poly (7) adenosine linker (HD22-7A-DAB) and characterized its thrombin inhibition activity using a fluorogenic substrate cleavage assay. The results reveal that HD22-7A-DAB has a much higher thrombin inhibition activity (IC50 = 0.2 nM) than DAB (IC50 = 80 nM) or the unconjugated aptamer (IC50 > 1000 nM). The significantly higher inhibitory effect of HD22-7A-DAB compared to a 1:1 mixture of unconjugated DAB and HD22 also indicates that conjugation is essential to create an effective synergistic inhibitor. Notably, in the presence of 2 µM complementary DNA to HD22, the effect of HD22-7A-DAB conjugate was drastically diminished (IC50 > 1000 nM), which indicates that both aptamer and small molecule in ADIC can be reversed using an antidote oligonucleotide. HD22-7A-DAB also shows a UFH-rivaling anticoagulation effect and antidote-reversible behavior in a series of in vitro clotting assays, including thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thus the development of a thrombin-targeting small molecule aptamer conjugate inhibitor yields a potent and reversible anticoagulant with clinically relevant anticoagulant activity. In addition, these studies describe a generalizable strategy for rapidly developing aptamer-small molecule active site inhibitor conjugates for generate other potent and reversible inhibitors of the other coagulation proteases. These potent and reversible anticoagulant aptamer-active site drug conjugates have the potential to become UFH substituents in surgeries to reduce the risk of UFH-associated bleeding, UFH-insensitivity and HIT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Di-Arginine Additives for Dissociation of Gold Nanoparticle Aggregates: A Matrix-Insensitive Approach with Applications in Protease Detection.

We report the reversible aggregation of gold nanoparticles (AuNPs) assemblies via a di-arginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that the HS-PEG thiol group has a higher affinity for the AuNP surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies' dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNP suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates, or even seawater. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favored. Moreover, starting with AuNPs already aggregated ensured the absence of a background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNP assemblies and solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluids. Trypsin was chosen as the model enzyme, and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrates. The increase of positive charge of the HS-PEG-peptide conjugate quenched the dissociation capacity of the HS-PEG molecules, which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine.

Modulation of PC1/3 activity by a rare double-site homozygous mutation.

Preprotein convertase 1/3 deficiency is a rare autosomal recessive disorder in which patients present with malabsorptive diarrhea and a series of symptoms of endocrine disorders such as polydipsia, reactive hypoglycemia, growth hormone deficiency, hypothyroidism, adrenal insufficiency, and early onset obesity. In its essence, pituitary hormone deficiency is caused by insufficient cleavage of pituitary prohormones. Here, we describe a female child with a rare double-site homozygous mutation in PCSK1 (Proprotein convertase subtilisin/kexin-type 1) gene, and thereby intend to investigate the relationship between these novel mutation sites and changes in protein synthesis and function. We tested this patient's blood and urine fecal indicators of infection, blood electrolytes, and relevant endocrine hormone levels in the laboratory. Next Generation Sequencing was applied to screen the patient's DNA. Western Blot was performed to evaluate the mutant protein's expression. The enzymatic activity was measured as the rate of cleavage of a synthetic fluorogenic substrate in a specific solution. We found that this patient presented shortly after birth with uncorrectable diarrhea and symptoms of metabolic acidosis with hypothyroidism. Next Generation Sequencing revealed that a rare double-site homozygous missense mutation, c.763G > A (p.G255R) and c.758C > T (p.S253L), were detected in exon 7 of PCSK1 (Proprotein convertase subtilisin/kexin-type 1) gene on chromosome 5 of the patient. Western blotting revealed that there was no significant decrease in protein synthesis levels in the mutant phenotype compared to the wild type. Compared with WT type, the proteins expressed by the mutations showed a significant decrease in the enzyme activity towards the fluorescent substrates. However, neither the single site mutation p.S253L or p.G255R, nor the double-site mutation of both, all showed no significant differences from each other. These two missense mutations have not been reported before, and it is even rarer to find homozygous variation of two sites in one patient. This study identifies two novel mutations for the first time and further investigates the changes in protein synthesis and enzyme activity, providing a new pathway to continue to explore the pathogenesis of diseases associated with the function of PC1/3.

Pd nanocubes supported on SiW12 @Co-ZIF Nanosheets for High-efficiency rupture of ether bonds in model and actual lignin

A rational design of the intrinsic lattice structure of catalysts is vitally important to address catalytic performance. Herein is reported a top-down synthesis for the fabrication of a bifunctional metallic catalyst. First, palladium nanocubes with (100) lattices planes were synthesized through a shape-control strategy; next, the as-synthesized Pd/Co-ZIF was used as a template for in-situ phase reconstruction by a spontaneous phase transformation process in the presence of H4SiW under solvothermal conditions. Ultimately, formation of 3D hierarchical flower-like bimetallic nitride nanosheets (SiW12 @Pd/Co-ZIF-NS) were generated because of this process. The SiW12 @Pd/Co-ZIF-NS catalyst not only displays excellent catalytic transfer hydrogenolysis for C-O bond cleavage of lignin dimeric substrates, but also maintains superior recycling durability due to an encapsulation phenomenon preventing aggregation. Finally, the resulting nanocomposites exhibit superior activity and good size selectivity toward catalytic transfer hydrogenolysis of the actual lignin substrates to produce a significant amount of low-molecular-weight aromatic compounds.

PreAcrs: a machine learning framework for identifying anti-CRISPR proteins

BackgroundAnti-CRISPR proteins are potent modulators that inhibit the CRISPR-Cas immunity system and have huge potential in gene editing and gene therapy as a genome-editing tool. Extensive studies have shown that anti-CRISPR proteins are essential for modifying endogenous genes, promoting the RNA-guided binding and cleavage of DNA or RNA substrates. In recent years, identifying and characterizing anti-CRISPR proteins has become a hot and significant research topic in bioinformatics. However, as most anti-CRISPR proteins fall short in sharing similarities to those currently known, traditional screening methods are time-consuming and inefficient. Machine learning methods could fill this gap with powerful predictive capability and provide a new perspective for anti-CRISPR protein identification.ResultsHere, we present a novel machine learning ensemble predictor, called PreAcrs, to identify anti-CRISPR proteins from protein sequences directly. Three features and eight different machine learning algorithms were used to train PreAcrs. PreAcrs outperformed other existing methods and significantly improved the prediction accuracy for identifying anti-CRISPR proteins.ConclusionsIn summary, the PreAcrs predictor achieved a competitive performance for predicting new anti-CRISPR proteins in terms of accuracy and robustness. We anticipate PreAcrs will be a valuable tool for researchers to speed up the research process. The source code is available at: https://github.com/Lyn-666/anti_CRISPR.git.

Identification of Molecular Determinants in iRhoms1 and 2 That Contribute to the Substrate Selectivity of Stimulated ADAM17.

The metalloprotease ADAM17 is a key regulator of the TNFα, IL-6R and EGFR signaling pathways. The maturation and function of ADAM17 is controlled by the seven-membrane-spanning proteins iRhoms1 and 2. The functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ, in that stimulated shedding of most ADAM17 substrates tested to date can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of very few ADAM17 substrates, such as TGFα. The first transmembrane domain (TMD1) of iRhom2 and the sole TMD of ADAM17 are important for the stimulated shedding of ADAM17 substrates by iRhom2. However, little is currently known about how the iRhoms interact with different substrates to control their stimulated shedding by ADAM17. To provide new insights into this topic, we tested how various chimeras between iRhom1 and iRhom2 affect the stimulated processing of the EGFR-ligands TGFα (iRhom1- or 2-dependent) and EREG (iRhom2-selective) by ADAM17. This uncovered an important role for the TMD7 of the iRhoms in determining their substrate selectivity. Computational methods utilized to characterize the iRhom1/2/substrate interactions suggest that the substrate selectivity is determined, at least in part, by a distinct accessibility of the substrate cleavage site to stimulated ADAM17. These studies not only provide new insights into why the substrate selectivity of stimulated iRhom2/ADAM17 differs from that of iRhom1/ADAM17, but also suggest new approaches for targeting the release of specific ADAM17 substrates.

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly–Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2–P1↓P1’ (Gly–Gly↓Ala) position using the YESS–PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly–Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1–P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1’ position Ala of Smt3 was found to play a vital role in maintaining Ulp1’s precise cleavage after the Gly–Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Comparative effectiveness of the new calpain inhibitor NPO-2270 versus enalapril in pressure overload-induced heart failure

Abstract Background Studies performed in multiple preclinical models support the contribution of the Ca2+-dependent cysteine proteases calpains to ventricular remodelling and heart failure (HF). However, pharmacological calpain inhibition has not yet been tested in patients with HF mainly due to the limitations of available inhibitors. Purpose To determine the effect of NPO-2270 (NPO), a new ketoamide derivative calpain inhibitor, in a mouse model of pressure-overload and compare its effectiveness with that of enalapril. Methods C57BL6 mice were subjected to transverse aortic constriction (TAC) for 4 weeks. Mice were randomised to receive orally administered NPO or enalapril at the dose of 10 mg/kg/day once a day, or vehicle, starting at day 7 after TAC surgery. The combination of both drugs and the effect of NPO starting 1 day after surgery were tested in additional TAC mice. Echocardiographic data, markers of hypertrophy, fibrosis, calpain activity and cleavage of calpain substrates were measured at different time points. Results TAC increased calpain-1 and -2 expression and activity. Administration of NPO and enalapril prevented the progression of hypertrophy and interstitial fibrosis induced by TAC with no statistically significant differences between the two treatments. However, ongoing ventricular dysfunction was less severe in the NPO group than in the enalapril group (27% of LVEF reduction in control group, 6% in NPO group and 16% in the enalapril group after TAC, P=0.024 between NPO and enalapril groups). The combined treatment or the administration of NPO from the first day after TAC surgery was not superior to NPO alone starting 7 days after TAC. These differences in LVEF correlated with better preservation of cadherin-based cell adhesion complex in mice treated with NPO-2270. No adverse effects associated with long-term NPO administration were observed in a sham group. Conclusions The new calpain inhibitor NPO-2270 prevents the development of hypertrophy and fibrosis with similar efficacy than enalapril but prevents cardiac dysfunction more effectively in a preclinical model of pressure overload when given orally at equivalent doses. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III of the Spanish Ministry of Health

A biodegradable and cofactor self-sufficient aptazyme nanoprobe for amplified imaging of low-abundance protein in living cells

Despite the progress on the analysis of proteins either in vitro or in vivo, detection and imaging of low-abundance proteins in living cells still remains challenging. Herein, a novel biodegradable and cofactor self-sufficient DNAzyme nanoprobe has been deve-loped for catalytic imaging of protein in living cells with signal amplification capacity. This DNAzyme nanoprobe is constructed by assembling a DNAzyme subunit-containing aptamer hairpin (HP), another DNAzyme subunit strand (DS), and the molecular beacon (MB) substrate strand onto pH-sensitive ZnO@polydopamine nanorods (ZnO@PDA NRs) that work as DNAzyme cofactor suppliers. Such a nanoprobe can facilitate cellular uptake of DNA molecules and protection of them from nuclease degradation as well as release of them in cells by lysosomal acid-triggered dissolution of ZnO@PDA NRs into Zn2+ as DNAzyme cofactor. Upon recognition and binding with the intracellular protein target, the stem of HP is opened, after which the opened HP hybridizes with DS and generates activated DNAzymes. Each activated DNAzyme can catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers, resulting in the separation of the quenched fluorophore/quencher pair labeled in MB and the generation of significantly amplified fluorescence. Using nucleolin (NCL) as a model protein, this nanoprobe enables the analysis of NCL with a detection limit of 1.8 pM, which are at least two orders of magnitude lower than that of non-catalytic imaging probe. Moreover, it could accurately distinguish tumor cells and normal cells by live cell NCL imaging. And the experimental results are also further verified by flow cytometry assays. The developed nanoprobe can be easily extended to detect other biomolecules by the change of their corresponding aptamer sequences, thus providing a promising tool for highly sensitive imaging of low-abundance biomolecules in living cells.

Expression, purification, and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.

Phycocyanin purified from Westiellopsis sp. induces caspase 3 mediated apoptosis in breast cancer cell line MDA-MB-231

Phycocyanin is a water-soluble blue pigment biliprotein that has been utilized as a nutritional supplement for its potent antioxidant and anti-inflammation activity. In this study, three cyanobacterial species from the North-Eastern India, Calothrix sp. (BTA-476), Calothrix sp. (BTA-857), and Westiellopsis sp. (BTA-801) are taxonomically and molecularly identified using 16s rRNA gene sequencing. The cyanobacteria with the highest phycocyanin content were chosen, and phycocyanin was extracted. The purity was determined to be 2.3, which was almost 5 times purer than the crude extract, with a recovery rate of 47.47 %. Purified phycocyanin has strong cytotoxic potential with high anti-proliferative activity in breast cancer cell line MDA-MB-231, resulting in chromatin condensation and the formation of apoptotic bodies. Apoptotic cell death was validated by caspase 3 activation where phycocyanin IC50 120 μg mL−1 is equivalent to 31.37 nM AMC caspase 3 cleavage of DEVD-AMC substrate.

A peptide-DNA hybrid bio-nanomicelle and its application for detection of caspase-3 activity.

Bio-nanomicelles based on biomaterials such as nucleic acids, peptides, glycans, and lipids have developed rapidly in the field of bioanalysis. Although DNA and peptides have unique advantages, unfortunately, there are few bio-nanomicelles integrating DNA with peptides. Here, we designed a peptide-DNA hybrid bio-nanomicelle for the activity detection of caspase-3. The detection mechanism is based on caspase-3 specific recognition and cleavage of peptide substrates, which owns high sensitivity and selectivity. Under optimal conditions, the detection of caspase-3 activity can be achieved using our designed bio-nanomicelles and the detection limit is 0.72 nM. Furthermore, the proposed method was also successfully applied for the detection of caspase-3 in cell lysate samples after apoptosis-inducing.

Biochemical Characterizations of the Putative Endolysin Ecd09610 Catalytic Domain from Clostridioides difficile.

Clostridioides difficile is the major pathogen of pseudomembranous colitis, and novel antimicrobial agents are sought after for its treatment. Phage-derived endolysins with species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified an endolysin gene, Ecd09610, which has an uncharacterized domain at the N-terminus and two catalytic domains that are homologous to glucosaminidase and endopeptidase at the C-terminus. Genes containing the two catalytic domains, the glucosaminidase domain and the endopeptidase domain, were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. The purified domain variants showed lytic activity almost specifically for C. difficile, which has a unique peptide bridge in its peptidoglycan. This species specificity is thought to depend on substrate cleavage activity rather than binding. The domain variants were thermostable, and, notably, the glucosaminidase domain remained active up to 100 °C. In addition, we determined the optimal pH and salt concentrations of these domain variants. Their properties are suitable for formulating a bacteriolytic enzyme as an antimicrobial agent. This lytic enzyme can serve as a scaffold for the construction of high lytic activity mutants with enhanced properties.

Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

Dual-miRNA-Propelled Three-Dimensional DNA Walker for Highly Specific and Rapid Discrimination of Breast Cancer Cell Subtypes in Clinical Tissue Samples

Highly sensitive α-hemolysin nanopore detection of MUC1 based on 3D DNA walker

Mucin 1(MUC1) is an effective marker of breast cancer, so it is of great significance to develop a simple, sensitive and highly selective MUC1 detection sensor. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of MUC1. The presence of MUC1 triggered the modification of the DNAzyme walking chain on the surface of Fe3O4 nanoparticles and separation from the aptamer. In the presence of Zn2+, DNAzyme catalyzed hydrolytic cleavage of the hairpin substrate at the scissile rA. The DNAzyme was divided into two fragments and ssDNA was released. ssDNA products from the hairpin substrate can generate a current blocking signal during α-hemolysin nanopore testing. The frequency of signature events showed a linear response toward the concentration of MUC1 in the range of 0.01 nM–100 nM. The sensing system also exhibited high selectivity against other protein and can be used for the detection of real sample.

Features of the hemostatic system at combat-caused acoustic injuries

Introduction: Exposure to high-intensity sounds can cause significant damage to the human auditory system. Joint damage to the auditory system by high-intensity sound and shock waves causes acubarotrauma. Such lesions are systemic in nature, affecting not only the auditory system but also the entire human body. It significantly impairs the quality of life and general condition of the body, causes numerous complications. Therefore, the issues of diagnosis and treatment of hearing impairments caused by acutraumas and acubarotraumas received in combat conditions are highly relevant. Aim: to study the hemostatic indices of patients with combat-caused acoustic trauma and identify the most informative ones. Patients and methods: 22 patients with combat acoustic trauma and 10 healthy volunteers were tested. The object of the study was citrate plasma, which was obtained by standard methods. The content of fibrinogen was determined by thrombin-like enzyme Ancistron-H. Partially activated thromboplastin time and prothrombin time were determined by tests of Siemens, USA. The share of functionally inactive forms of prothrombin was determined by Ecamulin-based diagnostic test. The content of protein C activity was determined by cleavage of synthetic chromogenic substrate (Siemens, USA). The concentrations of soluble fibrin and D-dimer were evaluated by immunodiagnostic test systems "DIA-soluble fibrin" and "DIA-D-dimer" (Diaprof-Med, Ukraine). Statistical processing of the results was performed by WinPEPI package for biometric statistical processing with Student's t-test for evaluation of the difference between patients and the control group. Results and discussion: It was found that fibrinogen’s level in 25% of patients remained at the level of control. In 27% of patients this index decreased on the level of the trend, and in 50% - increased 1.6 times. The elevat ion of fibrinogen’s level proves for a risk of thrombotic complications, since this acute phaseprotein is well-known index of hypercoagulability. Partially activated thromboplastin time index in 14% of the tested group remained at the level of control values. In 14% of patients it was probably 1.4 times shorter, and in 73% of patients it was 1.6 times longer than control. Prolonged partially activated thrombin time may prove for the increased level of circulating coagulation factor VIII. In 82% of patients the index of prothrombin time was probably 2.4 times prolonged, where as in 18% of patients was almost at the level of control. It may prove for an imbalance between the components of blood clotting system. The indices of Ecamulin-based test were multidirectional. In 18% of patients it was on the level of control, in 55% of patients it was slightly shorter, and in 27% of patients - probably elongated 1.4 times. The level functionally inactive forms of prothrombin in 50% of patients didn’t differ of control values, in 23% it was tended to decrease, and in 27% it was 1.6 more. The latter proves for the activation of the blood coagulation system. Protein C is a physiological inhibitor of the blood coagulation system. Its’ level in 50% of patients was reduced by 2.2 times in contrast to control, in 14% it remained on the normal level. As well-known, a decrease in protein C level proves for imbalance between coagulation and anticoagulation in the direction of the first one and is associated with thrombosis. The content of D-dimer in 33% of patients was on the level of control, while the level of soluble fibrin was increased 4 times. In 42% there was a 1.4-fold decrease in D-dimer content at a slight increase of soluble fibrin level. In 25% of patients the indices of D-dimer and soluble fibrin were increased in 4 and 7 times, respectively. Elevated level of soluble fibrin is an early prognostic index of the activation of blood clotting system. Comprehensive determination of the content of soluble fibrin and the level of D-dimer allows to estimate the correlation between the accumulation and destruction of the soluble fibrin. Conclusion: From the above data it follows that the determination of the single index is not enough for adequate assess of the risk of thrombosis and the effectiveness of antithrombotic therapy. On this reason the comprehensive definition of the noted indices should be included into the clinical practice a mandatory algorithm for the diagnosis of prothrombotic conditions in patients with acoustic trauma hearing disturbances.

Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening.

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.

Wash- and Separation-Free Electrochemical Detection of Proteases

Proteases play crucial roles in biological systems, including digestion, catabolism, and cell signaling. Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain) protease, has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine–proline–arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical–chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases.References S. Park, K. Park, H. S. Na, J. Jung, H. Yang, Anal. Chem. 93, 5644 (2021) S. Park, J. Shin, J. Kwon, W. Lee, J. Kim, G. Kim, J. M. Joo, H. Yang, ACS Sens. 6, 1305 (2021) S. Park, G. Kim, J. Seo, H. Yang, Anal. Chem. 88, 11995 (2016)S. Park, Y. M. Shin, J. Seo, J.-J. Song, H. Yang, Analyst 141, 2481 (2016)S. Park, Y. Shin, J.-J. Song, H. Yang, Biosens. Bioelectron. 72, 211 (2015)S. Park, H. Yang, Analyst, 139, 4051 (2014)