Specific Cleavage Research Articles

Smartphone and handheld fluorometer enable rapid point of care testing of Escherichia coli in urinary tract infections via specific proteolytic cleavage and cascade amplifications

Culture-free, rapid, and ultrasensitive quantification of Escherichia coli (E. coli) is urgently needed to diagnose clinical urinary tract infections (UTIs), especially point-of-care testing (POCT). Herein, a homogeneous sensitive multi-modal (fluorometer, smartphone, and self-developed handheld fluorometer) assay was developed to detect E. coli within 30 min, and nanoscale luminescent cadmium telluride quantum dots (CdTe QDs) were employed as signal reporters. E. coli outer membrane protease T (OmpT) is a biomarker for the specific proteolytic cleavage of peptides. p-Aminophenol (PAP), generated by leucine aminopeptidase (LAP) via secondary cleavage, was used to reduce Ag+. Cascade amplifications included Ag+ and copper sulfide nanoparticles (CuS NPs) to release Cu2+, CdTe QDs, and Cu2+/CuS NPs, which can ensure detection sensitivity. The method achieved single bacterium detection with great stability and selectivity. The fluorometer, handheld fluorometer and smartphone red-green-blue (RGB) value-based modes were determined to be 50, 15, and 15 clinical urine samples, respectively. The findings were consistent with clinical culture counting, and an area under the curve (AUC) of 0.95 with 90% sensitivity and 100% specificity was achieved. Overall, this OmpT-based catalytic biosensor can provide a solution for the POCT diagnosis of UTIs based on E. coli detection.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts.

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

Single-Cell Caspase-3 Measurement Using a Biomimetic Nanochannel.

Direct single-cell caspase-3 (Casp-3) analysis has remained challenging. A study of single-cell Casp-3 could contribute to revealing the fundamental pathogenic mechanisms in Casp-3-associated diseases. Here, a biomimetic nanochannel capable of single-cell sampling and ionic detection of intracellular Casp-3 is devised, which is established upon the installment of target-specific organic molecules (luc-DEVD) within the orifice of a glass nanopipette. The specific cleavage of luc-DEVD by Casp-3 could induce changes of inner-surface chemical groups and charge properties, thus altering the ionic response of the biomimetic nanochannel for direct Casp-3 detection. The practical applicability of this biomimetic nanochannel is confirmed by probing intracellular Casp-3 fluctuation upon drug stimulation and quantifying the Casp-3 evolution during induced apoptosis. This work realizes ionic single-cell Casp-3 analysis and provides a different perspective for single-cell protein analysis.

First-in-human validation of enzymolysis clearance strategy for decreasing renal radioactivity using modified [68Ga]Ga-HER2 Affibody.

Enzymolysis clearance strategy, characterized by releasing the non-reabsorbable radioactive fragment under the specific cleavage of enzymes, is confirmed to be a safe and effective way to reduce the renal radioactivity accumulation in mice. However, the effectiveness of this strategy in humans remains unknown. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various types of tumors, and radiolabeled HER2 Affibody is believed to be an attractive tool for HER2-targeted theranostics. However, its wide application is limited by the high and persistent renal uptake. In this study, we intend to validate the effectiveness of enzymolysis clearance strategy in reducing renal accumulation by using a modified HER2 Affibody. A new HER2 Affibody ligand, NOTA-MVK-ZHER2:2891, containing a cleavable Met-Val-Lys (MVK) linker was synthesized and labeled with 68Ga. The microPET imaging study was performed in SKOV-3 tumor mice to assess the uptakes of the control ligand and the MVK one in tumors and kidneys. Seven healthy volunteers were included for biodistribution and dosimetric studies with both the control and MVK ligands performed 1week apart. Urine and blood samples from healthy volunteers were collected for in vivo metabolism study of the two ligands. Four HER2-positive and two HER2-negative patients were recruited for [68Ga]Ga-NOTA-MVK-ZHER2:2891 PET/CT imaging at 2 and 4h post-injection (p.i.). [68Ga]Ga-NOTA-MVK-ZHER2:2891 was stable both in PBS and in mouse serum. MicroPET images showed that the tumor uptake of [68Ga]Ga-NOTA-MVK-ZHER2:2891 was comparable to that of [68Ga]Ga-NOTA-ZHER2:2891 at all the time points, while the kidney uptake was significantly reduced 40min p.i. (P < 0.05). The biodistribution study in healthy volunteers showed that the kidney uptake of MVK ligand was significantly lower than that of the control ligand at 1h p.i. (P < 0.05), with the SUVmean of 34.3 and 45.8, respectively, while the uptakes of the two ligands in the other organs showed negligible difference. The effective doses of the MVK ligand and the control one were 26.1 and 28.7 µSv/MBq, respectively. The enzymolysis fragment of [68Ga]Ga-NOTA-Met-OH was observed in the urine samples of healthy volunteers injected with the MVK ligand, indicating that the enzymolysis clearance strategy worked in humans. The PET/CT study of patients showed that the range of SUVmax of HER2-positive lesions was 9.4-21, while that of HER2-negative lesions was 2.7-6.2, which suggested that the MVK modification did not affect the ability of ZHER2:2891 structure to bind with HER2. We for the first time demonstrated that enzymolysis clearance strategy can effectively reduce renal radioactivity accumulation in humans. This strategy is expected to decrease renal radiation dose of peptide and small protein-based radiotracers, especially in the field of radionuclide therapy.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14–57%) and proteins (9–31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE–PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

Massively parallel profiling of RNA-targeting CRISPR-Cas13d

CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d reveals that it does not require a protospacer flanking sequence but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is penalized by mismatches in the distal crRNA-target RNA region, while alterations in the proximal region inhibit nuclease activity. A biophysical model built from these data reveals that target recognition initiates in the distal end of the target RNA. Using this model, we design crRNAs that can differentiate between SARS-CoV-2 variants by modulating nuclease activation. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme.

Development of a tag-free plant-made interferon gamma production system with improved therapeutic efficacy against viruses.

Plants offer a promising platform for cost-effective production of biologically active therapeutic glycoproteins. In previous studies, we have developed a plant expression system based on Bamboo mosaic virus (BaMV) by incorporating secretory signals and an affinity tag, which resulted in notably enhanced yields of soluble and secreted fusion glycoproteins (FGs) in Nicotiana benthamiana. However, the presence of fusion tags on recombinant glycoproteins is undesirable for biomedical applications. This study aimed to develop a refined expression system that can efficiently produce tag-free glycoproteins in plants, with enhanced efficacy of mature interferon gamma (mIFNγ) against viruses. To accommodate the specific requirement of different target proteins, three enzymatically or chemically cleavable linkers were provided in this renovated BaMV-based expression system. We demonstrated that Tobacco etch virus (TEV) protease could process the specific cleavage site (LTEV) of the fusion protein, designated as SSExtHis(SP)10LTEV-mIFNγ, with optimal efficiency under biocompatible conditions to generate tag-free mIFNγ glycoproteins. The TEV protease and secretory-affinity tag could be effectively removed from the target mIFNγ glycoproteins through Ni2+-NTA chromatography. In addition, the result of an antiviral assay showed that the tag-free mIFNγ glycoproteins exhibited enhanced biological properties against Sindbis virus, with comparable antiviral activity of the commercialized HEK293-expressed hIFNγ. Thus, the improved BaMV-based expression system developed in this study may provide an alternative strategy for producing tag-free therapeutic glycoproteins intended for biomedical applications.

CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone mRNA processing.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options, potentiating the importance of uncovering novel drug targets. Here, we target cleavage and polyadenylation specificity factor 3 (CPSF3), the 3' endonuclease that catalyzes mRNA cleavage during polyadenylation and histone mRNA processing. We find that CPSF3 is highly expressed in PDAC and is associated with poor prognosis. CPSF3 knockdown blocks PDAC cell proliferation and colony formation in vitro and tumor growth in vivo. Chemical inhibition of CPSF3 by the small molecule JTE-607 also attenuates PDAC cell proliferation and colony formation, while it has no effect on cell proliferation of nontransformed immortalized control pancreatic cells. Mechanistically, JTE-607 induces transcriptional readthrough in replication-dependent histones, reduces core histone expression, destabilizes chromatin structure, and arrests cells in the S-phase of the cell cycle. Therefore, CPSF3 represents a potential therapeutic target for the treatment of PDAC.

ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

Structural and mechanistic insights into ribosomal ITS2 RNA processing by nuclease-kinase machinery.

Precursor ribosomal RNA (pre-rRNA) processing is a key step in ribosome biosynthesis and involves numerous RNases. A HEPN (higher eukaryote and prokaryote nucleotide binding) nuclease Las1 and a polynucleotide kinase Grc3 assemble into a tetramerase responsible for rRNA maturation. Here, we report the structures of full-length Saccharomyces cerevisiae and Cyberlindnera jadinii Las1-Grc3 complexes, and C. jadinii Las1. The Las1-Grc3 structures show that the central coiled-coil domain of Las1 facilitates pre-rRNA binding and cleavage, while the Grc3 C-terminal loop motif directly binds to the HEPN active center of Las1 and regulates pre-rRNA cleavage. Structural comparison between Las1 and Las1-Grc3 complex exhibits that Grc3 binding induces conformational rearrangements of catalytic residues associated with HEPN nuclease activation. Biochemical assays identify that Las1 processes pre-rRNA at the two specific sites (C2 and C2'), which greatly facilitates rRNA maturation. Our structures and specific pre-rRNA cleavage findings provide crucial insights into the mechanism and pathway of pre-rRNA processing in ribosome biosynthesis.

Structural and mechanistic insights into ribosomal ITS2 RNA processing by nuclease-kinase machinery

Precursor ribosomal RNA (pre-rRNA) processing is a key step in ribosome biosynthesis and involves numerous RNases. A HEPN (higher eukaryote and prokaryote nucleotide binding) nuclease Las1 and a polynucleotide kinase Grc3 assemble into a tetramerase responsible for rRNA maturation. Here, we report the structures of full-length Saccharomyces cerevisiae and Cyberlindnera jadinii Las1-Grc3 complexes, and C. jadinii Las1. The Las1-Grc3 structures show that the central coiled-coil domain of Las1 facilitates pre-rRNA binding and cleavage, while the Grc3 C-terminal loop motif directly binds to the HEPN active center of Las1 and regulates pre-rRNA cleavage. Structural comparison between Las1 and Las1-Grc3 complex exhibits that Grc3 binding induces conformational rearrangements of catalytic residues associated with HEPN nuclease activation. Biochemical assays identify that Las1 processes pre-rRNA at the two specific sites (C2 and C2′), which greatly facilitates rRNA maturation. Our structures and specific pre-rRNA cleavage findings provide crucial insights into the mechanism and pathway of pre-rRNA processing in ribosome biosynthesis.

Thrombomodulin Switches Signaling and Protease-Activated Receptor 1 Cleavage Specificity of Thrombin.

Cleavage of the extracellular domain of PAR1 (protease-activated receptor 1) by thrombin at Arg41 and by APC (activated protein C) at Arg46 initiates paradoxical cytopathic and cytoprotective signaling in endothelial cells. In the latter case, the ligand-dependent coreceptor signaling by EPCR (endothelial protein C receptor) is required for the protective PAR1 signaling by APC. Here, we investigated the role of thrombomodulin in determining the specificity of PAR1 signaling by thrombin. We prepared a PAR1 knockout (PAR1-/-) EA.hy926 endothelial cell line by CRISPR/Cas9 and transduced PAR1-/- cells with lentivirus vectors expressing PAR1 mutants in which either Arg41 or Arg46 was replaced with an Ala. Furthermore, human embryonic kidney 293 cells were transfected with wild-type or mutant PAR1 cleavage reporter constructs carrying N-terminal Nluc (NanoLuc luciferase) and C-terminal enhanced yellow fluorescent protein tags. Characterization of transfected cells in signaling and receptor cleavage assays revealed that, upon interaction with thrombomodulin, thrombin cleaves Arg46 to elicit cytoprotective effects by a β-arrestin-2 biased signaling mechanism. Analysis of functional data and cleavage rates indicated that thrombin-thrombomodulin cleaves Arg46>10-fold faster than APC. Upon interaction with thrombin, the cytoplasmic domain of thrombomodulin recruited both β-arrestin-1 and -2 to the plasma membrane. Thus, the thrombin cleavage of Arg41 was also cytoprotective in thrombomodulin-expressing cells by β-arrestin-1-biased signaling. APC in the absence of EPCR cleaved Arg41 to initiate disruptive signaling responses like thrombin. These results suggest that coreceptor signaling by thrombomodulin and EPCR determines the PAR1 cleavage and signaling specificity of thrombin and APC, respectively.

Characteristics of the a sequence of the duck Plague virus genome and specific cleavage of the viral genome based on the a sequence

During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

Mn2+-induced structural flexibility enhances the entire catalytic cycle and the cleavage of mismatches in prokaryotic argonaute proteins.

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid-based biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn2+ bound in the pockets, as verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn2+ in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn2+ in solution enhances the flexibility of diverse pAgo proteins. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn2+, leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn2+-induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicate that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, Mn2+-induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn2+ in solution, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.

Next-generation CRISPR/Cas-based ultrasensitive diagnostic tools: current progress and prospects.

CRISPR/Cas has been explored as a powerful molecular scissor that uses a double-strand break mediated non-homologous end joining (NHEJ) or homology-directed repair (HDR) to achieve precise gene editing. Cas effectors come in several different forms, each with its own set of features and applications. SpCas9 was the first and most extensively studied CRISPR/Cas version, and it has been hailed as a biotechnology breakthrough that could potentially correct mutations to treat genetic diseases. Recently, the Cas12 and Cas13 effector variants of Class II, Type V and Type VI, have been explored for their specific collateral cleavage (trans-cleavage) activity on target recognition. This trans-cleavage activity helps in the recognition of target nucleic acids. CRISPR diagnostics technology utilized the binding of crRNA with Cas12/13 protein to form the Ribonucleoproteins (RNPs) complex, which further cleaves the target sequence in cis-cleavage, followed by the activation of trans-cleavage of a nonspecific fluorescent DNA/RNA probe, resulting in the production of a fluorescent signal that could be quantitatively recorded. Later, nanotechnology and mobile-based detection applications were incorporated into the system to develop advanced lateral flow-based strips and are also associated with the technology to make it more feasible. Overall, this review compiles the experimental evidence consolidating the application of CRISPR/Cas as next-generation biosensors for diagnostic applications.

SpecLipIDA: a pseudotargeted lipidomics approach for polyunsaturated fatty acids in milk.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), play an important role in the nutritional value of milk lipids. However, a comprehensive analysis of PUFAs and their esters in milk is still scarce. In this study, we developed a novel pseudotargeted lipidomics approach, named SpecLipIDA, for determining PUFA lipids in milk. Triglycerides (TGs) and phospholipids (PLs) were separated using NH2 cartridges, and mass spectrometry data in the information-dependent acquisition (IDA) mode were preprocessed by MS-DIAL, leading to improved identification in subsequent targeted analysis. The target matching algorithm, based on specific lipid cleavage patterns, demonstrated enhanced identification of PUFA lipids compared to the lipid annotations provided by MS-DIAL and GNPS. The approach was applied to identify PUFA lipids in various milk samples, resulting in the detection of a total of 115 PUFA lipids. The results revealed distinct differences in PUFA lipids among different samples, with 44 PUFA lipids significantly contributing to these differences. Our study indicated that SpecLipIDA is an efficient method for rapidly and specifically screening PUFA lipids.

An ATMND/SGI based three-way junction ratiometric fluorescent probe for rapid and sensitive detection of bleomycin.

In this work, we developed a rapid and sensitive label-free ratiometric fluorescent (FL) probe for the detection of bleomycin (BLM). The probe consists of a DNA sequence (D6) and two fluorophore groups, 2-amino-5,6,7-trimethyl-1,8-naphthalene (ATMND) and SYBR Green I (SGI). The D6 sequence could be folded into a three-way junction structure containing a C-C mismatch position in the junction pocket. The unique "Y" structure not only could entrap ATMND in the mismatch pocket with high affinity, leading to FL quenching at 408 nm, but also embed SGI in the grooves of the double-stranded portion, resulting in FL enhancement at 530 nm. In the presence of BLM-Fe(II), the "Y" structure of D6 was destroyed due to the specific cleavage of the BLM recognition site, the 5'-GT-3' site in D6. This caused the release of ATMND and SGI and thus the ratiometric signal change of FL enhancement by ATMND and FL quenching by SGI. Under optimal conditions, the ratiometric probe exhibited a linear correlation between the intensity ratio of F408/F530 and the concentration of BLM in the range of 0.5-1000 nM, with a detection limit of 0.2 nM. In addition, the probe was applied to detect BLM in human serum samples with satisfactory results, indicating its good clinical application potential.

Enhanced Expression and Bioactivity Assessment of Recombinant SUMO‐Protease‐1 in E. coli BL21 (DE3) via Cleavage of His6‐SMT3‐SDF‐1 Fusion Protein

Background. The SUMO fusion system is advantageous in improving the solubility and correct folding of proteins that are difficult to produce. SUMO‐protease‐1 (Ulp1) is a key enzyme in this system, and its proper expression and purification in Escherichia coli BL21 (DE3) are critical to its efficiency. Objective. To optimize Ulp1 expression and purification using His6‐SUMO‐SDF‐1 as a model, aiming to improve efficiency across similar fusion proteins. Methods. We provided a codon‐optimized Ulp1 gene into the pET28a vector. Essential culture factors, including IPTG concentration, incubation length of time, and temperature, were precisely adjusted. Ulp1 was purified using Ni‐NTA affinity chromatography. A cleavage activity assay of recombinant Ulp1 is carried out using the His6‐SUMO‐SDF‐1 fusion protein to demonstrate how well the optimized settings work. Furthermore, the activity of recombinant SDF‐1 was evaluated using the scratch wound healing assay. Results. Ulp1 expression was optimized in E. coli using the pET28a vector, with optimum conditions including 0.1 mM IPTG, 28°C incubation temperature, and an overnight period of time. Recombinant Ulp1 was purified using Ni‐NTA affinity chromatography, resulting in high purity. Enzymatic activity was shown by the effective cleavage of the His6‐SUMO‐SDF‐1 fusion protein. In the scratch wound‐healing assay, recombinant SDF‐1 increased cell migration following cleavage, confirming that biological activity was retained. These findings illustrate Ulp1’s efficient synthesis and effectiveness for prospective applications in biology. Conclusion. This study successfully showed the expression and purification of the recombinant Ulp1 enzyme, showing the most suitable growing conditions for productive protein production. Ulp1’s functionality was validated by its specific cleavage of the His6‐SUMO‐SDF‐1 fusion protein, as well as the fragmented SDF‐1 purified and its biological activity in promoting cell migration. These findings highlight Ulp1’s potential in protein processing as well as its application in biotechnological and biological research.

Ultrasensitive electrochemical detection and inhibition evaluation of DNA methyltransferase based on cascade strand displacement amplification.

An electrochemical sensing approach for ultrasensitive DNA methyltransferase (MTase) activity assay is proposed. After specific cleavage reaction in the presence of a methylated state, strand displacement polymerization (SDP) is initiated in the solution. The product of upstream SDP further triggers downstream SDP, which enriches abundant electrochemical species at the electrode. The whole process is quite convenient with shared enzymes. Due to the cascade signal amplification, ultrahigh sensitivity is promised. Inhibitor screening results are also demonstrated to be good. Besides, target MTase can be accurately determined in human serum samples, confirming excellent practical utility. This work provides a reliable approach for the analysis of MTase activity, which is of vital importance for related biological studies and clinical applications.

TsRNA: A Promising Biomarker in Breast Cancer.

tRNA-derived small RNAs (tsRNAs) are a novel class of non-coding small RNAs, generated from specific cleavage sites of tRNA or pre-tRNA. tsRNAs can directly participate in RNA silencing, transcription, translation, and other processes. Their dysregulation is closely related to the occurrence and development of various cancers. Breast cancer is one of the most common and fastest-growing malignant tumors in humans. tsRNAs have been found to be dysregulated in breast cancer, serving as a new target for exploring the pathogenesis of breast cancer. They are also considered new tumor markers, providing a basis for diagnosis and treatment. This article reviews the generation, classification, mechanism of action, function of tsRNAs, and their biological effects and related mechanisms in breast cancer, in the hope of providing a new direction for the diagnosis and treatment of breast cancer.