Specific Cleavage Research Articles

Proteolysis of host DEAD-box RNA helicase by potyviral proteases activates plant immunity.

The precise mechanisms by which plant viral proteases interact with and cleave host proteins, thereby participating in virus-host interactions, are not well understood. Potyviruses, the largest group of known plant-infecting RNA viruses, are known to rely on the nuclear inclusion protease a (NIa-Pro) for the processing of viral polyproteins. Here, we demonstrate that the proteolytic activity of NIa-Pro from potyvirus turnip mosaic virus (TuMV) is indispensable for inducing hypersensitive cell death in Nicotiana benthamiana. NIa-Pro targets and degrades the host DEAD-box protein 5 (DBP5) via a specific cleavage motif, which initiates host cell death. Both the silencing of DBP5 and the overexpression of NIa-Pro lead to an increased frequency of stop codon readthrough, which could be potentially harmful to the host, as it may result in the production of aberrant proteins. Unlike the NIa-Pro of most other potyviruses, the NIa-Pro of tobacco etch virus can also degrade DBP5 and trigger cell death, in both pepper and N. benthamiana. Furthermore, we discovered that the TuMV-encoded nuclear inclusion b can counteract NIa-Pro-induced cell death by co-opting DBP5. These findings unveil hitherto uncharacterized roles for plant virus proteases in cleaving host proteins and highlight the role of host DBP5 in modulating plant immunity.

A DNA sensor based on CbAgo effector protein and on a dual electrochemical signal amplification strategy for B19 parvovirus detection

Human parvovirus B19 is a prevalent childhood infectious virus that poses a great challenge to public health, so the detection of B19V is of great importance. In this study, a DNA sensor based on CbAgo, a Cas effector, and a dual electrochemical signal amplification strategy was developed by using a novel nanocomposite MnO2/CMK-3/g-C3N4/AgNPs for initial signal amplification, with CMK being an ordered mesoporous carbon nanomaterial. Single-walled carbon nanotubes (SWCNTs) were used as electrocatalytic probes for secondary signal amplification to detect B19 DNA. The detection process begins with polymerase chain reaction (PCR) amplification using the B19V infectious clone plasmid (pB19-M20) as a template and NS1-F/R as primers, followed by specific cleavage of B19 DNA based on the programmable cutting sites of CbAgo effector protein. This study enriches the application of Argonaute proteins in sensing and introduces a novel method to detect B19V. Under optimized conditions, the biosensor can detect B19 DNA in the range of 10−15–10−10 M, with a detection limit (LOD) of 0.2 fM. The results indicate that the developed DNA sensor holds promise for reliable and sensitive detection of B19 DNA in human serum.

A simple colorimetric detection of telomerase exploiting specific cleavage of exonuclease III coupled with telomeric DNA controlled aggregation of nanogold.

Telomerase activity has piqued scientists' interest for the reason that it has the potential to be employed for early-stage cancer detection, anticancer therapy and studies related to cancer progression and metastasis. Several approaches have been developed to detect telomerase activity. However, these approaches were lengthy, challenging to quantify, of limited sensitivity and prone to polymerase chain reaction (PCR)-related artefacts. We herein developed a novel nanoplasmonic sensing platform for colorimetric detection of telomerase activity relying on the telomere elongation of telomerase at the 3' end, structure-specific cleavage activity of exonuclease III that removes mononucleotides from the 3'-hydroxyl termini of double-stranded DNA, and electrostatic interaction of elongated telomeres with plasmonic nanoparticles. Using HeLa cells as a model for colorimetric detection of telomerase activity, this biosensor could detect telomerase activity with a detection limit of ∼100 cells per reaction by visible inspection and ∼5 cells per reaction by spectroscopic measurement and analysis time within about three hours. The proposed sensing method provided a novel tool for simple, rapid, and low-cost detection of telomerase activity, eliminating the necessity for thermal cycling, primers in PCR-based assays, and amplification of telomerase extension products. It exhibits significant potential as a label-free, simple, ultrasensitive strategy for on-site detection of telomerase activity in proteomics and clinical diagnostics.

Enhancement of foaming performance of hempseed protein by limited enzymatic hydrolysis: From the viewpoint of the structural and interfacial rheological attributes

Effects of limited enzymolysis by Alcalase and Protamex on the foaming performance of hempseed protein (HPI) and its correlation with structural and interfacial rheological characteristics were investigated. Proteolysis induced a conformational shift from α-helix and β-sheet to random coil, indicating enhanced molecular flexibility. The surface hydrophobicity of Alcalase hydrolysates encountered an initial increase (5 min) followed by a sharp drop with prolonged hydrolysis, whereas Protamex showed minimal effects. Both proteases reduced total sulfhydryl content and caused a redshift in intrinsic fluorescence, particularly Alcalase. The specific cleavage pattern of Alcalase generated peptides with pronouncedly higher solubility (up to 47.0 %) relative to Protamex. The flexible conformation and increased solubility induced by moderate proteolysis, notably with Alcalase, facilitated viscoelastic interfacial membranes with consolidated intermolecular interactions, consequently contributing to more homogeneous and smaller bubble structures. Optimal foaming capacity (95.0 %) and foam stability (90.9 %) were achieved with Alcalase for 20 min and 5 min, respectively.

Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.

The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min-1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.

Postproline Cleaving Enzymes also Show Specificity to Reduced Cysteine.

In proteomics, postproline cleaving enzymes (PPCEs), such as Aspergillus niger prolyl endopeptidase (AnPEP) and neprosin, complement proteolytic tools because proline is a stop site for many proteases. But while aiming at using AnPEP in online proteolysis, we found that this enzyme also displayed specificity to reduced cysteine. By LC-MS/MS, we systematically analyzed AnPEP sources and conditions that could affect this cleavage preference. Postcysteine cleavage was blocked by cysteine modifications, including disulfide bond formation, oxidation, and alkylation. The last modification explains why this activity has remained undetected so far. In the same experimental paradigm, neprosin mimicked this cleavage specificity. Based on these findings, PPCEs cleavage preferences should be redefined from post-Pro/Ala to post-Pro/Ala/Cys. Moreover, this evidence demands reconsidering PPCEs applications, whether cleaving Cys-rich proteins or assessing Cys status in proteins, and calls for revisiting the proposed enzymatic mechanism of these proteases.

Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations

CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.

A distinctive function of GH13_8 subfamily glycogen branching enzyme in Anaerococcus prevotii DSM 20548: Preference to create very short branches

Glycogen branching enzymes (GBEs; EC 2.4.1.18) are essential for forming α-1,6-O-glycosidic branches in starch modification and glycogen biosynthesis. They are classified into glycoside hydrolase (GH) families 13 and 57. GH13 GBEs are further divided into subfamilies GH13_9, containing predominantly sequences from bacteria, and GH13_8, comprising sequences from both bacteria and eukaryotes. So far, only three eukaryotic GH13_8 enzymes have been studied in detail while no crystal structures or functional activities of prokaryotic GH13_8 GBEs have been reported. In this study, the GH13_8 and GH13_9 GBE of Anaerococcus prevotii (Ap) were studied in detail. It was shown for the first time that this prokaryotic GH13_8 GBE is active on amylose and creates α-1,6-O-glycosidic linked branches. In contrast to GH13_9 GBEs, the ApGBE13_8 is active on very short oligosaccharides ranging from DP2 to DP5 (degree of polymerization) transferring glucose or maltose. The preference for short oligosaccharides might be correlated with the presence of two short beta stranded loops at position 131 and 509. These loops may function like a ‘door,’ dynamically adjusting to the donor chain, affecting branch length and cleavage specificity. These findings reveal ApGBE13_8's distinct function, advance GH13_8 research, and suggest potential applications for GH13_8 GBEs in starch modification.

Comprehensive protease specificity profiling.

Protease enzymes are of great importance in medicine, industry, and as research tools. Despite the crucial need for detailed knowledge of their proteolytic cleavage specificity, many proteases are poorly characterized. We present a method for fully characterizing the cleavage specificity of proteases through the comprehensive profiling of all possible permutations of octamer peptide substrates in a single experiment. The powerful combination of in vitro selection with high-throughput sequencing, mass spectrometry, and automated motif mining enabled the screening of mixtures of >10 12 unique peptides. We developed freely available software that easily integrates the massive amounts of cleavage data into user-friendly specificity information. We applied this method to three different proteases that had either narrow (factor Xa) or broad specificity (ADAM17 and streptopain). The resulting specificity maps revealed motifs that corroborate canonical known cleavage sites, yet step further into extended spectrum preferences and yield insights into the function of broad specificity proteases.

Functional Analysis of CPSF30 in Nilaparvata lugens Using RNA Interference Reveals Its Essential Role in Development and Survival.

The brown planthopper (Nilaparvata lugens) is a major pest threatening global rice production, significantly reducing yields annually. As N. lugens increasingly develops resistance to conventional control methods, such as chemical pesticides, there is an urgent need for innovative and sustainable pest management strategies. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a key protein involved in mRNA 3' end processing, yet its function in N. lugens remains poorly understood. This study aims to elucidate the role of CPSF30 in the growth and development of N. lugens and evaluate its potential as a target for RNA interference (RNAi)-based pest control strategies. We cloned and characterized the cDNA sequence of NlCPSF30, which encodes a protein of 341 amino acids containing five CCCH zinc-finger domains and two CCHC zinc-knuckle domains. Sequence alignment revealed that NlCPSF30 is highly conserved among insect species, particularly in the zinc-finger domains essential for RNA binding and processing. Phylogenetic analysis showed that NlCPSF30 is closely related to CPSF30 proteins from other hemipteran species. Expression analysis indicated that NlCPSF30 is most highly expressed in the fat body and during the adult stage, with significantly higher expression in females than in males. RNAi-mediated silencing of NlCPSF30 in third-instar nymphs resulted in severe phenotypic abnormalities, including disrupted molting and increased mortality following injection of double-stranded RNA (dsRNA) targeting NlCPSF30. Moreover, it influenced the expression of genes associated with hormone regulation, namely NlHry, NlE93, and NlKr-h1. These results suggest that NlCPSF30 is integral to critical physiological processes, with its disruption leading to increased mortality. Our findings identify NlCPSF30 as an essential gene for N. lugens' survival and a promising target for RNAi-based pest management strategies. This study provides a valuable molecular target and theoretical insights for developing RNAi-based control methods against N. lugens.

Sulfated peptides: key players in plant development, growth, and stress responses.

Peptide hormones regulate plant development, growth, and stress responses. Sulfated peptides represent a class of proteins that undergo posttranslational modification by tyrosylprotein sulfotransferase (TPST), followed by specific enzymatic cleavage to generate mature peptides. This process contributes to the formation of various bioactive peptides, including PSKs (PHYTOSULFOKINEs), PSYs (PLANT PEPTIDE CONTAINING SULFATED TYROSINE), CIFs (CASPARIAN STRIP INTEGRITY FACTOR), and RGFs (ROOT MERISTEM GROWTH FACTOR). In the past three decades, significant progress has been made in understanding the molecular mechanisms of sulfated peptides that regulate plant development, growth, and stress responses. In this review, we explore the sequence properties of precursors, posttranslational modifications, peptide receptors, and signal transduction pathways of the sulfated peptides, analyzing their functions in plants. The cross-talk between PSK/RGF peptides and other phytohormones, such as brassinosteroids, auxin, salicylic acid, abscisic acid, gibberellins, ethylene, and jasmonic acid, is also described. The significance of sulfated peptides in crops and their potential application for enhancing crop productivity are discussed, along with future research directions in the study of sulfated peptides.

DNA polymerase mediated CRISPR/Cas12a trans-cleavage for dual-mode quantification of uracil DNA glycosylase activity

Since an unusual expression of uracil-DNA glycosylase (UDG) is often associated with the pathogenesis of numerous disorders, the detection of UDG activity is regarded as a promising application in disease diagnosis. Here, we develop a DNA polymerase-mediated CRISPR/Cas12a trans cleavage strategy, which can achieve dual-mode determination of UDG activity. By introducing a hairpin DNA probe containing a single uracil base, the probe undergoes specific cleavage and elongation under the existence of UDG only, thus activating the trans cleavage of ssDNA regardless of its length and sequence. To accommodate different detection modes, the ssDNA was further modified by fluorophore-quencher pairs or designed for connecting magnetic microparticles (MMPs) and polystyrene microparticles (PMPs). Finally, the UDG activity is quantified by fluorescence signal and microparticle accumulation length on a microfluidic chip visible to the naked eye. This strategy provides a detectable minimum UDG concentration of 0.00047 U/mL for fluorescent mode and 0.0048 U/mL for microfluidic mode. Furthermore, we performed the UDG inhibition test and detected UDG activity in cell lysates, suggesting its potential for inhibitor screening and detection of UDG in biological samples.

Development of an on-site real-time dual detection method for norovirus and rotavirus using RPA-CRISPR/Cas12,13

Norovirus (NoV) and rotavirus (RoV) are the two most prevalent foodborne viruses responsible for infectious gastroenteritis, with high contamination rates in fruits and vegetables. Monitoring and accurately identifying these pathogens are crucial for preventing human foodborne illnesses. Therefore, developing a rapid, highly specific, and sensitive detection method for NoV and RoV is essential. An on-site real time dual detection method for Noroviruses of genogroup I (GI-NoV) and Rotavirus of group A (A-RoV) was established by integrating Recombinase Polymerase Amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) system. The DNA amplification products generated by multiplex RPA were simultaneously detected by Cas12a and Cas13a in a single tube. Utilizing the trans-cleavage preferences of Cas12a and Cas13a, the specific cleavage of DNA and RNA probes carrying different fluorescent groups within the reaction system was achieved. The results indicated that the established dual detection method had a detection limit of 102 copies/μL for GI-NoV and A-RoV. Specificity assay showed the established method only detected GI-NoV and A-RoV, with no cross-reactivity with other four foodborne viruses. Furthermore, the detection capability of this method was validated in strawberries and lettuce. The method enables the simultaneous detection of GI-NoV and A-RoV in fresh produce at 37 °C within 60 min. Detection results were interpreted by measuring fluorescence values or observing color changes under blue light at the end of the reaction. This technique required minimal instrumentation, provided excellent visual representation of results, and served as a technical reference for the rapid on-site detection of foodborne viruses in food matrices.

Divergent molecular assembly and catalytic mechanisms between bacterial and archaeal RNase P in pre-tRNA cleavage

Ribonuclease P (RNase P) plays a vital role in the maturation of tRNA across bacteria, archaea, and eukaryotes. However, how RNase P assembles various components to achieve specific cleavage of precursor tRNA (pre-tRNA) in different organisms remains elusive. In this study, we employed single-molecule fluorescence resonance energy transfer to probe the dynamics of RNase P from E. coli (Escherichia coli) and Mja (Methanocaldococcus jannaschii) during pre-tRNA cleavage by incorporating five Cy3-Cy5 pairs into pre-tRNA and RNase P. Our results revealed significant differences in the assembly and catalytic mechanisms of RNase P between E. coli and Mja at both the RNA and protein levels. Specifically, the RNA of E. coli RNase P (EcoRPR) can adopt an active conformation that is capable of binding and cleaving pre-tRNA with high specificity independently. The addition of the protein component of E. coli RNase P (RnpA) enhances and accelerates pre-tRNA cleavage efficiency by increasing and stabilizing the active conformation. In contrast, Mja RPR is unable to form the catalytically active conformation on its own, and at least four proteins are required to induce the correct folding of Mja RPR. Mutation experiments suggest that the functional deficiency of Mja RPR arises from the absence of the second structural layer, and proper intermolecular assembly is essential for Mja RNase P to be functional over a broad temperature range. We propose models to illustrate the distinct catalytic patterns and RNA-protein interactions of RNase P in these two organisms.

RPA-CRISPR-Cas-Mediated Dual Lateral Flow Assay for the Point-of-Care Testing of HPV16 and HPV18.

The incidence of cervical cancer caused by human papillomavirus (HPV) infection has increased in recent years. More than half of all cervical cancer cases are due to HPV16 and HPV18 infection, so HPV16 and HPV18 testing is essential to prevent cervical cancer. HPV testing is mainly carried out in hospitals, but it is subject to time and specialized medical facilities. On the other hand, home self-testing using simple diagnostics would present an attractive alternative due to privacy and flexibility with regard to time and place, provided sufficient sensitivity and specificity can be achieved. In this work, a dual lateral flow assay based on RPA-CRISPR-Cas12a/13a (named RC-LFA) for HPV detection was described. Taking advantage of the cleavage specificity of Cas12a and Cas13a, a CRISPR-Cas12a/Cas13a system was designed to detect HPV16 and HPV18. The lateral flow strip with two test lines was designed to suit the CRISPR-Cas12a/Cas13 system. RC-LFA achieves rapid and simultaneous detection of HPV16 and HPV18 with high specificity and sensitivity (10 copies/μL) in about 40 min from the extraction of nucleic acid to an instrument-free readout. RC-LFA is user-friendly and instrument-free, making it a promising method for HPV self-tests at home.

Clean and Complete Protein Digestion with an Autolysis Resistant Trypsin for Peptide Mapping.

Peptide mapping requires cleavage of proteins in a predictable fashion so that target protein-specific peptides can be reliably identified and quantified. Trypsin, a commonly used protease in this process, can also undergo self-cleavage or autolysis, thereby reducing the effectivity and even cleavage specificity at lysine and arginine residues. Here, we report highly efficient and reproducible peptide mapping of biotherapeutic monoclonal antibodies. We highlight the properties of a homogeneous chemically modified trypsin on thermal stability, a 54% increase in melting temperature with an 84% increase in energy required for unfolding, an indication of more thermally stable trypsin, >90% retained intact mass peak area after exposure to digestion conditions confirming autolysis resistance, 10× more intensity for intact enzyme compared to trypsin of similar source and narrower molecular weight distribution with LC-MS indicative of low degradation compared to 3 other types of trypsin. Finally, we show the utility of this autolysis-resistant trypsin in characterizing biotherapeutic monoclonal antibodies consistently and reliably showing a >30% reduction in missed cleavage for a short-duration protein digestion time of 30 min compared to heterogeneously modified trypsin of a similar source.

12231 PAPP-A As A Potential Target In Thyroid Eye Disease

Abstract Disclosure: C.A. Conover: None. L.K. Bale: None. D. Hamadi: None. M. Stan: ; Tourmaline, Genentech, Third Rock Ventures, ArgenX, Septerna, OrthoDi. ; Horizon, Immunovant, Lassen, Sling. Background: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Studies revealed an interaction between thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor-I receptor (IGF-IR) that is essential in promoting TED pathogenesis. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. PAPP-A is metalloprotease that can increase pericellular IGF bioavailability through specific cleavage of inhibitory IGF binding proteins, in particular IGFBP-4. We propose that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitory antibodies attenuates IGF-IR signaling in TED with fewer side effects. Methods: Informed consent was obtained from TED patients undergoing surgery in Mayo Clinic operating suites in Rochester, MN. Retro-orbital tissue was collected from 19 TED patients and 2 control subjects for fibroblast isolation and culture and was performed in a sterile tissue culture facility. TED and control fibroblasts were treated with pro-inflammatory cytokines, and flow separation of CD34- and CD34+ orbital fibroblasts was done, the latter representing infiltrating fibrocytes into the orbit in TED. Main outcome measured were PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. Results: Primary cultured cells showed that PAPP-A is expressed in orbital fibroblasts from TED patients but not in control subject fibroblasts and were markedly increased by pro-inflammatory cytokines stimulation. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A’s proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. CD34+ orbital fibroblasts represent 80% of cells in culture from TED patients, and approximately 70% of these cells were accountable for PAPP-A and IGF-IR expression. Conclusion: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis. Presentation: 6/2/2024

Electrical and fluorescence in situ monitoring of tumor microenvironment-based pH-responsive polymer dot coated surface

A boronate-ester structure forming a pH-responsive polymer dot (Plu-PD) coated biosensor between carbonized-sp2 rich dopamine-alginate [PD(Alg)] and boronic acid-grafted Pluronic (BA-Pluronic) was developed for the electrochemical and fluorescence detection of cancer cells. The reduced fluorescence (FL) resulting from fluorescence resonance energy transfer (FRET) mediated by π-π interactions within Plu-PD was successfully reinvigorated through the specific cleavage of the boronate-ester bond, triggered by the acidic conditions prevailing in the cancer microenvironment. The anomalous variations in extracellular pH levels observed in cancer (pH ∼6.8), as opposed to the normal cellular pH range of approximately 7.4, serve as robust indicators for discerning cancer cells from their healthy counterparts. Moreover, the Plu-PD coated surface demonstrated remarkable adaptability in modulating its surface structure, concurrently exhibiting tunable electroconductivity under reduced pH conditions, thereby imparting selective responsiveness to cancer cells. The pH-modulated conductivity change was validated by a reduction in resistance from 211 ± 9.7 kΩ at pH 7.4 to 73.9 ± 9.4 kΩ and 61.5 ± 11.5 kΩ at pH 6.8 and 6.0, respectively. The controllable electrochemical characteristics were corroborated through in vitro treatment of cancer cells (HeLa, B16F10, and SNU-C2A) via LED experiments and wireless output analysis. In contrast, identical treatments yielded a limited response in normal cell line (CHO–K1). Notably, the Plu-PD coated surface can be seamlessly integrated with a wireless system to facilitate real-time monitoring of the sensing performance in the presence of cancer and normal cells, enabling rapid and accurate cancer diagnosis using a smartphone.

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication.

Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.

Specific cleavage of IGFBP-4 by papp-a in nervous tissue

Astrocytes are subtypes of glial cells involved in metabolic, structural, homeostatic, and neuroprotective processes that help neurons maintain viability. Insulin-like growth factors IGF-1 and IGF-2 are known to have neuroprotective effects on neurons and glial cells through interaction with specific receptors. IGF forms a complex with IGF-binding proteins (IGFBP) in nervous tissue and is released from the complex via IGFBP proteolysis by specific proteases. It has been reported that IGFBP-2, 5 and 6 are cleaved by specific proteases in the central nervous system (CNS), followed by IGF release; however, it was unknown whether IGFBP-4 was exposed to a particular proteolysis in nervous tissue. Using neurons and astrocytes derived from human induced pluripotent stem cell lines (hiPSC), as well as rat brain-sourced primary neuron-glia cultures, we demonstrated that IGFBP-4 is specifically cleaved in nervous tissue by the Pregnancy Associated Plasma Protein A (PAPP-A) protease and that this cleavage is IGF-dependent. Our results indicate that astrocyte rather than neuron PAPP-A cleaves IGFBP-4 in nervous tissue suggesting that this may be one of the fundamental mechanisms for IGF interchange between these two types of cells.