Inactive Fragments Research Articles

Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.

Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to early lethality in mice.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either as a homodimer, or as a more active BMP4/7 heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of BMP4 homodimers or heterodimers generated from BMP4, BMP4S91C or BMP4E93G precursor proteins in Xenopus embryos and found that these mutations reduce the activity of BMP4 homodimers but not heterodimers. We generated Bmp4 S91C and Bmp4 E93G knock-in mice and found that Bmp4 S91C/S91C mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart at E10.5. Most Bmp4 E93G/E93G mice die before weaning and Bmp4 -/E93G mutants die prenatally with reduced or absent eyes, heart and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from Bmp4 S91C and Bmp4 E93G embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because Bmp7 is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of cleaved ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.

The prodomain of bone morphogenetic protein 2 promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus. We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

The BMP2 prodomain promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers in vivo.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus . We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

Programmable RNA base editing with photoactivatable CRISPR-Cas13

CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.

Splittable systems in biomedical applications.

Splittable systems have emerged as a powerful approach for the precise spatiotemporal control of biological processes. This concept relies on splitting a functional molecule into inactive fragments, which can be reassembled under specific conditions or stimuli to regain activity. Several binding pairs and orthogonal split fragments are introduced by fusing with other modalities to develop more complex and robust designs. One of the pillars of these splittable systems is modularity, which involves decoupling targeting, activation, and effector functions. Challenges, such as off-target effects and overactivation, can be addressed through precise control. This review provides an overview of the design principles, strategies, and applications of splittable systems across diverse fields including immunotherapy, gene editing, prodrug activation, biosensing, and synthetic biology.

Split-Protein Therapeutic Platforms: Identifying Binder Pairs.

Therapeutic proteins, including enzymes, interferons, interleukins, and growth factors, are emerging as important modalities to treat many diseases that elude management by small molecule drugs. One challenge of protein treatment is the propensity for off-target or systemic activity. A promising approach to overcome such toxicity is to create conditionally active constructs by splitting the therapeutic protein into two, or more, inactive fragments and by fusing these fragments to binders (e.g., antibodies) that target distinct epitopes on a cell surface. When these antibodies bind to their respective targets, the protein fragments are brought into proximity and then reconstitute into the active form of the therapeutic protein. In this chapter, we describe approaches to determine antibody pairs that enable the reconstitution of the active protein. General computational and empirical methods are provided to facilitate the identification of pairs starting only from protein sequence data.

B-334 How to Prevent the Next Wave in Overdose Crisis?—Detect Designer Benzodiazepines Using CEDIA and DRI Assays

Abstract Background Benzodiazepines are a class of drugs used for relieving symptoms of anxiety, insomnia, agitation, muscle spasms, and alcohol withdrawal. Benzodiazepines are CNS depressants enhancing the effect of GABA neurotransmitter at GABA receptors and they are well-known for drug abuse potential and drug dependence.Designer benzodiazepines are new psychoactive substances emerging in the past two decades. Designer benzodiazepines are often taken by individuals who use hallucinogenic and stimulant drugs. The drug abuse can lead to respiratory depression, coma, or death. There exists a need to develop a new homogeneous enzyme immunoassay. Existing commercial immunoassay antibodies may also cross-react with designer benzodiazepines which have chemical structure closely related to classic therapeutic benzodiazepines. Herein, we evaluated designer benzodiazepines cross-reactivity using DRI Benzodiazepines and CEDIA Benzodiazepines assays and demonstrated the potential of detecting designer benzodiazepines in urines. Methods DRI assay is based on competition between drug labeled with glucose-6-phosphate dehydrogenase (G6PDH) and free drug in the urine sample for a fixed amount of antibody binding sites. The enzyme activity is determined spectrophotometrically at 340 nm by measuring its ability to convert NAD to NADH. CEDIA technology is based on the bacterial enzyme β-galactosidase which has been genetically engineered into two inactive fragments, Enzyme Acceptor (EA) and Enzyme Donor (ED). The enzyme activity is then determined spectrophotometrically at 570 nm. Designer Benzodiazepines tested in this study are 3-Hydroxyphenazepam, Adinazolam, Bromazolam, Clonazolam, Cloniprazepam, Deschloroetizolam, Diclazepam, Etizolam, Flubromazepam, Flubromazolam, Flunitrazolam, Meclonazepam, N-Desmethylflunitrazepam, Nifoxipam, Nimetazepam, Nitrazolam, and Pyrazolam. Compounds were spiked into drug free urine and cross-reactivity was evaluated using DRI® Benzodiazepine Assay and CEDIA® Benzodiazepine Assays on the Beckman Coulter AU680. Results 14 out of 17 (82%) tested designer benzodiazepines showed high cross-reactivity (>100%) in DRI benzodiazepine assay. 12 out of 17 (71%) tested designer benzodiazepines showed high cross-reactivity (>100%) in CEDIA assays with 200 ng/mL cutoff and 300 ng/mL cutoff. Compared to NPS-benzodiazepines trend data, this study demonstrated that DRI and CEDIA benzodiazepine assays can detect more than 50% (10 out of 19) designer benzodiazepines over 2020 Q1 to 2022 Q4 in the United States. Conclusion DRI and CEDIA Benzodiazepine Assays showed high cross reactivity of most seventeen designer benzodiazepines. The results provide potential tools for detect emerging designer benzodiazepines. Note: This was preliminary data that has not been reviewed by any regulatory authorities/cleared by FDA.

Tackling Tissue Macrophage Heterogeneity by SplitCre Transgenesis.

Macrophages represent a broad spectrum of distinct, but closely related tissue-resident immune cells. This presents a major challenge for the study of functional aspects of these cells using classical Cre recombinase-mediated conditional mutagenesis in mice, since single promoter-driven Cre transgenic models often display limited specificity toward their intended target. The advent of CRISPR/Cas9 technology has now provided a time- and cost-effective method to explore the full potential of binary transgenic, intersectional genetics. Specifically, the use of two promoters driving inactive Cre fragments that, when co-expressed, dimerize and only then gain recombinase activity allows the characterization and manipulation of genetically defined tissue macrophage subpopulations. Here, we will elaborate on the use of this protocol to capitalize on these recent technological advances in mouse genetics and discuss their strengths and pitfalls to improve the study of tissue macrophage subpopulations in physiology and pathophysiology.

Identification of Dipeptidyl-Peptidase 4 (DPP-4) Inhibitors from Miracle Berry Fruit (Synsepalum dulcificum) Extract

Dipeptidyl-peptidase 4 is a novel targeted enzyme in type 2 diabetes mellitus (T2DM) therapy due to its regulatory effect on incretin; glucagon-like peptide 1 (GLP-1) that responsible for stimulating insulin and suppressing glucagon secretion. DPP-4 degrades GLP-1 to biologically inactive fragments. In incretin-based therapies, DPP-4 inhibitor helps to restrain the inactivation of GLP-1, which in turn prolonging GLP-1’s half-life in blood and subsequently reducing the blood sugar more effectively. Nowadays, researchers are focusing to replace the synthetic drugs with natural-based drugs since they exert less toxicity. In our attempt to discover the natural inhibitor for DPP-4, miracle berry fruit was sequentially extracted with different solvents such as hexane, dichloromethane, chloroform, ethyl acetate and methanol and screened for the DPP-4 inhibitory activity. The analyses on the extractions show dichloromethane yielded the highest number of phenolic compounds with 38.37 mg GAE/g, while the highest number of flavonoids was found in hexane extract with 142.269 mg QE/g. On the other hand, qualitative analyses showed that methanol was able to extract alkaloids, terpenoids, saponins, tannins, quinones and carbohydrate from miracle berry fruit. It was also found that ethyl acetate extract was able to completely inhibit the enzyme. For further identification of constituent responsible for DPP-4 inhibitory activity, ethyl acetate extract was then subjected to silica gel column chromatography and eluted with different solvents with increasing polarity. The active fraction was then subjected to LC-MS/MS analysis and the results show it contains astragalin, nialamide, n-acetyl-l-phenylalanine, (+)-6-gingerol, 9-nitro oleate and 4(3H)-quinazolinone. The study indicates that miracle berry fruit extract contains natural DPP-4 inhibitor that can potentially be used for treating T2DM.

Negligible risk of surface transmission of SARS-CoV-2 in public transportation.

Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.

Investigation of Cyclo-Z Therapeutic Effect on Insulin Pathway in Alzheimer's Rat Model: Biochemical and Electrophysiological Parameters

Cyclo (his-pro-CHP) plus zinc (Zn+2) (Cyclo-Z) is the only known chemical that increases the production of insulin-degrading enzyme (IDE) and decreases the number of inactive insulin fragments in cells. The aim of the present study was to systematically characterize the effects of Cyclo-Z on the insulin pathway, memory functions, and brain oscillations in the Alzheimer's disease (AD) rat model. The rat model of AD was established by bilateral injection of Aβ42 oligomer (2,5nmol/10μl) into the lateral ventricles. Cyclo-Z (10mg Zn+2/kg and 0.2mg CHP/kg) gavage treatment started seven days after Aβ injection and lasted for 21 days. At the end of the experimental period, memory tests and electrophysiological recordings were performed, which were followed by the biochemical analysis. Aβ42 oligomers led to a significant increase in fasting blood glucose, serum insulin, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and phospho-tau-Ser356 levels. Moreover, Aβ42 oligomers caused a significant decrement in body weight, hippocampal insulin, brain insulin receptor substrate (IRS-Ser612), and glycogen synthase kinase-3 beta (GSK-3β) levels. Also, Aβ42 oligomers resulted in a significant reduction in memory. The Cyclo-Z treatment prevented the observed alterations in the ADZ group except for phospho-tau levels and attenuated the increased Aβ42 oligomer levels in the ADZ group. We also found that the Aβ42 oligomer decreased the left temporal spindle and delta power during ketamine anesthesia. Cyclo-Z treatment reversed the Aβ42 oligomer-related alterations in the left temporal spindle power. Cyclo-Z prevents Aβ oligomer-induced changes in the insulin pathway and amyloid toxicity, and may contribute to the improvement of memory deficits and neural network dynamics in this rat model.

Prognostic value of N-terminal Brain Natriuretic Peptide (NT-proBNP) in Risk Assessment of Adverse Cardiovascular Events in Patients with Atrial Fibrillation and Heart Failure with Reduced Left Ventricular Systolic Function

According to Russian epidemiological studies, the incidence of chronic heart failure (HF) in the general population is approximately 7%, increasing from 0.3% in the group aged 2029 years to 70% in patients aged 90 years [1]. In the general population, the incidence of atrial fibrillation (AF) ranges from 1% to 2%, which increases with age, that is, from 0.5% at the age of 4050 years to 5%15% at the age of 80 years [2]. HF and AF aggravate significantly each others course and mutually increase the risk of adverse outcomes [3, 4]. Moreover, the incidence of AF in patients with HF increases with increasing New York Heart Association (NYHA) grade; that is, among patients with HF of NYHA grade I, the incidence of AF is 5%, whereas among patients with HF NYHA grade IV, the AF incidence in 50% [5].
 Chronic HF is a syndrome with complex pathophysiology, which is characterized by the activation of neurohumoral systems, namely, the reninangiotensinaldosterone system (RAAS), sympathetic nervous system (SNS), and insufficient activity of the natriuretic peptide (NUP) system. In the early stage of HF, i.e. asymptomatic dysfunction of the left ventricle, the activation of the SNS and RAAS plays a compensatory role aimed at maintaining cardiac output and circulatory homeostasis [6]. Moreover, the NUP system has a counter-regulatory function in relation to the RAAS and SNS, and with prolonged and excessive activation of the SNS and RAAS or with insufficient NUP system activity, imbalance occurs and HF progresses [7].
 The brain natriuretic peptide (BNP) and biologically inactive N-terminal fragment of BNP (NT-proBNP) are the most studied and significant in clinical practice representatives of the NUP system. BNP and NT-proBNP are secreted by cardiomyocytes of the left ventricular (LV) myocardium in response to an increase in the mechanical load and stress of the LV myocardium. NT-proBNP is widely used as a test to rule out HF in patients with dyspnea. The NUP level also correlates with the severity and prognosis in patients with an established diagnosis of HF, and studies have reported that the NUP level acts as a criterion for treatment efficiency in patients with HF [8]. NT-proBNP is a biomarker not only for HF but also for several other conditions, such as acute coronary syndrome and myocardial infarction (MI), because it is associated with an increased risk of death from all causes, regardless of age, stable effort angina grade, myocardial infarction history, and LV ejection fraction (LVEF) [9].
 NT-proBNP levels can be influenced by several additional factors such as age, obesity, or glomerular filtration rate. The prognostic value of NT-proBNP is relevant in comorbid patients with AF associated HF because AF can increase NT-proBNP levels independently [10]. Given that NUP secretion depends on intracardiac hemodynamics, the NT-proBNP levels may also depend on the approach to managing AF. Tachycardia is associated with high NT-proBNP levels [11].
 The rhythm control approach has advantages over the heart rate control approach in patients with HF and LVEF 50% to reduce mortality and the number of unplanned hospitalizations due to HF progression [12].
 To date, the prognostic significance of NT-proBNP levels in relation to the risk of adverse events in patients with HF and reduced LV systolic function associated with AF, depending on the approach of AF management, remains unresolved.
 This study aimed to assess the predictive value of NT-proBNP in relation to the development of adverse cardiovascular events in patients with permanent or persistent AF associated with HF and LVEF 50%.

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction.

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACE-N). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling.

Identifying Protein-Protein Interactions by Proximity Biotinylation with AirID and splitAirID.

Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.

Optically activated proximity labeling for spatially and temporally resolved proteomics.

Membrane protein interactions are essential for the proliferation, differentiation and survival of cells. However, due to the lack of appropriate technologies, the interactomes of membrane proteins, at distinct spatial locations within the cell and at different stages of the cell cycle, are poorly understood. Here, we develop a light-activated proximity labeling method for high-precision mapping of membrane protein interactions with high spatial and temporal resolution. Our method combines a split version of the proximity biotinylation enzyme, TurboID, with the optically activated protein dimerization pair CRY2 and CIB1. As proof of principle, we engineer cells expressing CIB1 and an inactive TurboID fragment on the cell membrane along with the complementary TurboID fused to CRY2 in the cytoplasm. Using immunofluorescence imaging and mass spectrometry, we demonstrate that the split-TurboID fragments assemble, become functional and biotinylate proteins, only upon exposure to light. Next we tag E-cadherin, an essential cell-cell adhesion protein, with our optically activated proximity labeling system and validate it by comparing the measured interactome upon light exposure with known E-cadherin binding partners. Our results demonstrate that light-triggered proximity labeling can map the E-cadherin interactome with high precision. We anticipate that our optically activated technique will serve as a revolutionary tool to map junctional protein interactions within cells, with high fidelity, in a previously unprecedented, spatially and temporally resolved fashion.

Photodegradable antimicrobial agents: towards structure optimization.

Antibiotic resistance continues to be an ominous threat facing human health globally and urgent action is required to limit the loss of human life. The pollution of antibiotics into the environment is one of the drivers behind the crisis. With this in mind, we have developed novel photodecomposable antimicrobial agents based on an ethanolamine scaffold, which upon photoirradiation decomposes into two major inactive fragments. Herein we describe our further work on the synthesis of novel ethanolamines with a particular focus on structure activity relationship, resulting in four new active compounds which photodecomposed into inactive fragments.

Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates.

Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.

Soluble Complement Receptor 1 Therapeutics

Human Complement Receptor 1 (CR1/CD35) is a potent negative regulator of the complement system. Its mechanism of action is through interaction with the complement activation fragments, C3b and C4b to mediate decay acceleration of the C3 and C5 convertase complexes as well as cleavage of both ligands into inactive fragments via cofactor activity. The result is inhibition of the classical, lectin, and alternative complement pathways. This article will focus on recombinant soluble forms of CR1 that have been generated as potential therapeutics for complement-mediated disorders. Specifically, we will review and contrast the in vitro and in vivo properties of: sCR1 (BRL55730/TP10/CDX-1135), the soluble full-length extracellular domain of human CR1; sCR1-sLex (TP20), a glyco-engineered version of sCR1 additionally targeted to activated endothelium; APT070 (Mirococept), a CR1 fragment conjugated to a myristoylated peptide to enhance tissue targeting; and CSL040, a soluble truncated version of the CR1 extracellular domain which exhibits altered potency and pharmacokinetic properties as compared to the parental molecule. The data obtained from studies on the effects of these CR1-based molecules in animal models of disease and their therapeutic applications will also be discussed.

TEspeX: consensus-specific quantification of transposable element expression preventing biases from exonized fragments.

SummaryTransposable elements (TEs) play key roles in crucial biological pathways. Therefore, several tools enabling the quantification of their expression were recently developed. However, many of the existing tools lack the capability to distinguish between the transcription of autonomously expressed TEs and TE fragments embedded in canonical coding/non-coding non-TE transcripts. Consequently, an apparent change in the expression of a given TE may simply reflect the variation in the expression of the transcripts containing TE-derived sequences. To overcome this issue, we have developed TEspeX, a pipeline for the quantification of TE expression at the consensus level. TEspeX uses Illumina RNA-seq short reads to quantify TE expression avoiding counting reads deriving from inactive TE fragments embedded in canonical transcripts.Availability and implementationThe tool is implemented in python3, distributed under the GNU General Public License (GPL) and available on Github at https://github.com/fansalon/TEspeX (Zenodo URL: https://doi.org/10.5281/zenodo.6800331).Supplementary information Supplementary data are available at Bioinformatics online.