C-terminal Research Articles

Electrochemical biosensor based on Novozym 435 enzymatic ring-opening polymerization for CEA detection

Carcinoembryonic antigen (CEA) is a broad-spectrum tumour marker, with its concentration levels serving as a crucial predictor of cancer diagnosis. In this study, we synthesized a polycaprolactone (PCL) macromolecular polymer via enzyme ring-opening polymerization (eROP). This polymer was then utilized as a signal amplification element in the “sandwich structure” electrochemical impedance biosensor designed for CEA detection. Initially, 3-mercaptopropionic acid (MPA) was self-assembled onto the electrode surface via a gold-sulfur bond. The carboxyl terminus of MPA was then activated using carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Subsequently, antibody 1 (Ab1) was immobilised on the electrode surface through the formation of an amide bond, serving as a recognition probe. To prevent non-specific binding, the remaining sites were blocked with Bovine Serum Albumin (BSA). Following the specific capture of CEA by Ab1, the unreacted amino group on the electrode surface was sealed using acrolein. Antibody-2 (Ab2) was then introduced to specifically recognize CEA, forming a classic antibody-antigen–antibody “sandwich structure.” Finally, a DMPA (2,2-dihydroxymethylpropionic acid)-PCL (Polycaprolactone) polymer was conjugated to the electrode surface as a signal amplification unit. The impedance signal strength was subsequently measured using Electrochemical Impedance Spectroscopy (EIS). Under optimal conditions, the biosensor demonstrated a wide linear range (1 pg mL−1 ∼ 100 ng mL−1) and a low detection limit of 0.38 pg mL−1. The sensor also exhibited high selectivity, stability and reproducibility when tested with clinical serum samples, highlighting its potential applications for early diagnosis and clinical monitoring.

Insights into the regulation of CHIP E3 ligase-mediated ubiquitination of neuronal protein BNIP-H

Abstract BCL2/adenovirus E1B 19-kDa protein-interacting protein 2 homolog (BNIP-H or Caytaxin), pivotal adaptor protein that facilitates cerebellar cortex growth and synaptic transmission, is post-translationally modified to regulate neuronal function. This study reports the ubiquitination of BNIP-H by CHIP (Carboxyl terminus of Hsc70-Interacting Protein), a U-box containing E3 ligase that is also regulated via autoubiquitination. Specifically, it was observed that CHIP autoubiquitinated itself primarily at Lys23 and Lys31 in-vitro. Mutation of these residues show the autoubiquitination of successive lysines of CHIP. In total, nine lysines on CHIP were identified as the autoubiquitination sites, the collective mutation of which completely terminated its autoubiquitination. Additionally, CHIP-mediated ubiquitination of BNIP-H is completely inhibited when BNIP-H bears arginine mutations at four key lysine residues. Next, using hydrogen deuterium exchange mass spectrometry a model of a plausible mechanism was proposed. The model suggests transient N-terminal interactions between the CHIP and BNIP-H which allows for the swinging of U-Box domain of CHIP to ubiquitinate BNIP-H. Following complex dissociation, BNIP-H population is regulated via the ubiquitin-proteasome pathway. Collectively, these results aid in our understanding of CHIP-mediated BNIP-H ubiquitination and provide further insight into the roles of these proteins in neuritogenesis and neurotransmission.

Structural insights into regulated intramembrane proteolysis by the positive alginate regulator MucP from Pseudomonas aeruginosa

Regulated intramembrane proteolysis (RIP) is a fundamentally conserved mechanism involving sequential cleavage by a membrane-bound Site-1 protease (S1P) and a transmembrane Site-2 protease (S2P). In the opportunistic pathogen Pseudomonas aeruginosa, the alternate sigma factor σ22 activates alginate production and in turn is regulated by the MucABCD system. The anti-sigma factor MucA, which inhibits σ22, is sequentially cleaved via RIP by AlgW (S1P) and MucP (S2P) respectively. In this study, we report high-resolution crystal structures of the MucP PDZ1 and PDZ2 domains. Structural and binding analysis confirms that MucP PDZ2 recognizes the carboxy-terminal Ala136 residue of MucA following Site-1 cleavage by AlgW, while the peptide binding groove of PDZ1 is obstructed by a short α-helix. A structure of MucP PDZ2 with bound MucA peptide shows how PDZ2 binds the newly exposed carboxyl terminus of MucA following AlgW cleavage. The ability of a ΔmucP strain of P. aeruginosa to form biofilms was reduced to a similar extent as a ΔalgW strain. This work paves the way for further studies of MucP and other PDZ-containing S2Ps in regulated intramembrane proteolysis.

Cryptochrome magnetoreception: Time course of photoactivation from non-equilibrium coarse-grained molecular dynamics

Magnetoreception, the ability to sense magnetic fields, is widespread in animals but remains poorly understood. The leading model links this ability in migratory birds to the photo-activation of the protein cryptochrome. Magnetic information is thought to induce structural changes in cryptochrome via a transient radical pair intermediate. This signal transduction pathway has been the subject of previous all-atom molecular dynamics (MD) simulations, but insights were limited to short timescales and equilibrium structures. To address this, we developed a non-equilibrium coarse-grained MD simulation approach, exploring cryptochrome’s photo-reduction over 20 replicates of 20 µs each. Our results revealed significant structural changes across the protein, with an overall time constant of 3 µs. The C-terminal (CT) region responded on a timescale of 4.7 µs, followed by the EEE-motif, while the phosphate binding loop (PBL) showed slower dynamics (9 µs). Network analysis highlighted direct pathways connecting the tryptophan tetrad to the CT, and distant pathways involving the EEE and PBL regions. The CT-dynamics are significantly impacted by a rearrangement of tryptophan residues in the central electron transfer chain. Our findings underscore the importance of considering longer timescales when studying cryptochrome magnetoreception and highlight the potential of non-equilibrium coarse-grained MD simulations as a powerful tool to unravel protein photoactivation reactions.

Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions.

Helicobacter pylori (H. pylori) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA-NT and cagA-NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection. Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori.

Genome-Wide Analysis of HECT E3 Ligases Members in Phyllostachys edulis Provides Insights into the Role of PeHECT1 in Plant Abiotic Stress Response.

Homology to E6-AP Carboxy Terminus (HECT) E3 ubiquitin ligases play pivotal roles in plant growth, development, and responses to abiotic stresses. However, the function of HECT genes in Phyllostachys edulis (P. edulis) remains largely uninvestigated. In this study, a comprehensive genome-wide analysis of the HECT E3 ubiquitin ligases gene family in P. edulis was conducted, aiming to elucidate its evolutionary relationships and gene expansion. Analysis of gene structure, conserved motifs and domains, and synteny genome regions were performed. Furthermore, cis-elements in HECT gene promoters that respond to plant hormones and environmental stresses were identified and corroborated by expression data from diverse abiotic stress conditions and hormone treatments. Based on the co-expression network of PeHECTs under cold and dehydration stresses, PeHECT1 was identified as a key candidate gene associated with abiotic stress tolerance. Overexpression of PeHECT1 in tobacco leaves significantly upregulated genes related to reactive oxygen species (ROS) detoxification and polyamine biosynthesis. Yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase (dual-LUC) assays suggested that the transcription factor ETHYLENE RESPONSE FACTOR 3 (PeERF3) bound to the dehydration-responsive element (DRE) of the promoter of PeHECT1 and activated its transcription activity. Phylogenetic analysis indicated that PeHECT1 in P. edulis exhibited a close association with the diploid herbaceous bamboo Olyra latifolia, followed by the divergence of rice and bamboo. In summary, this study enhances our comprehensive understanding of the HECT E3 ubiquitin ligases gene family in P. edulis and highlights the potential role of PeHECT1 in plant abiotic stress response.

Identification and characterization of 16 tripartite motif-containing proteins from Takifugu obscurus

Tripartite motif-containing (TRIM) proteins play important roles in apoptosis, development, autophagy, and innate immunity in vertebrates. In this study, a total of 16 TRIM genes were cloned and identified from obscure puffer Takifugu obscurus. Multiple alignment results showed that most of the deduced ToTRIM proteins contained three typical motifs, a really interesting new gene (RING) zinc-finger domain, one B-box, and a coiled-coil domain, which together formed the TRIM motif found in this large family of proteins. The carboxyl terminus of ToTRIMs is architecturally unique, with frequent examples being the ADP ribosylation factor-like domain, the B30.2 (PRY-SPRY) domain, the fibronectin type III domain, and the pleckstrin homology domain. Phylogenetic analysis revealed that 16 ToTRIMs were evolutionarily closely related to their counterparts in other selected vertebrates. Tissue distribution analysis showed that the mRNA transcripts of most ToTRIM genes were constitutively expressed in all tissues examined, with relatively high expression in immune tissues. After infection with Vibrio harveyi, the expression levels of 16 ToTRIM genes in the kidney and liver were significantly upregulated, and their responses over time varied. Taken together, our results suggested that ToTRIM genes are involved in the antibacterial immune responses of T. obscurus, which is expected to provide new insights into the functional characteristics of TRIMs in teleost fish.

The Bor1 elevator transport cycle is subject to autoinhibition and activation

Boron, essential for plant growth, necessitates precise regulation due to its potential toxicity. This regulation is achieved by borate transporters (BORs), which are homologous to the SLC4 family. The Arabidopsis thaliana Bor1 (AtBor1) transporter from clade I undergoes slow regulation through degradation and translational suppression, but its potential for fast regulation via direct activity modulation was unclear. Here, we combine cryo-electron microscopy, mutagenesis, and functional characterization to study AtBor1, revealing high-resolution structures of the dimer in one inactive and three active states. Our findings show that AtBor1 is regulated by two distinct mechanisms: an autoinhibitory domain at the carboxyl terminus obstructs the substrate pathway via conserved salt bridges, and phosphorylation of Thr410 allows interaction with a positively charged pocket at the cytosolic face, essential for borate transport. These results elucidate the molecular basis of AtBor1’s activity regulation and highlight its role in fast boron level regulation in plants.

Steviol rebaudiosides bind to four different sites of the human sweet taste receptor (T1R2/T1R3) complex explaining confusing experiments.

Sucrose provides both sweetness and energy by binding to both Venus flytrap domains (VFD) of the heterodimeric sweet taste receptor (T1R2/T1R3). In contrast, non-caloric sweeteners such as sucralose and aspartame only bind to one specific domain (VFD2) of T1R2, resulting in high-intensity sweetness. In this study, we investigate the binding mechanism of various steviol glycosides, artificial sweeteners, and a negative allosteric modulator (lactisole) at four distinct binding sites: VFD2, VFD3, transmembrane domain 2 (TMD2), and TMD3 through binding experiments and computational docking studies. Our docking results reveal multiple binding sites for the tested ligands, including the radiolabeled ligands. Our experimental evidence demonstrates that the C20 carboxy terminus of the Gα protein can bind to the intracellular region of either TMD2 or TMD3, altering GPCR affinity to the high-affinity state for steviol glycosides. These findings provide a mechanistic understanding of the structure and function of this heterodimeric sweet taste receptor.

Nanoparticle-Directed Antioxidant Therapy Can Ameliorate Disease Progression in a Novel, Diet-Inducible Model of Coronary Artery Disease.

Oxidative stress plays a crucial role in the pathogenesis of coronary artery disease. In cardiovascular research using murine models, the generation and maintenance of models with robust coronary arterial atherosclerosis has been challenging. We characterized a new mouse model in which the last 3 amino acids of the carboxyl terminus of the HDL (high-density lipoprotein) receptor (SR-B1 [scavenger receptor class B, member 1]) were deleted in a low-density lipoprotein receptor knockout (LDLR-/-) mouse model (SR-B1ΔCT/LDLR-/-) fed an atherogenic diet. We also tested the therapeutic effects of an oxidative stress-targeted nanoparticle in atherogenic diet-fed SR-B1ΔCT/LDLR-/- mice. The SR-B1ΔCT/LDLR-/- mice fed an atherogenic diet had occlusive coronary artery atherosclerosis, impaired cardiac function, and a dramatically lower survival rate, compared with LDLR-/- mice fed the same diet. As SR-B1ΔCT/LDLR-/- mice do not exhibit female infertility or low pup yield, they are far easier and less costly to use than the previously described SR-B1-based models of coronary artery disease. We found that treatment with the targeted nanoparticles improved the cardiac functions and corrected hematologic abnormalities caused by the atherogenic diet in SR-B1ΔCT/LDLR-/- mice but did not alter the distinctive plasma lipid levels. The SR-B1ΔCT/LDLR-/- mice developed diet-inducible, fatal atherosclerotic coronary artery disease, which could be ameliorated by targeted nanoparticle therapy. Our study provides new tools for the development of cardiovascular therapies.

A deep intronic splice-altering AIRE variant causes APECED syndrome through antisense oligonucleotide-targetable pseudoexon inclusion.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a life-threatening monogenic autoimmune disorder primarily caused by biallelic deleterious variants in the autoimmune regulator (AIRE) gene. We prospectively evaluated 104 patients with clinically diagnosed APECED syndrome and identified 17 patients (16%) from 14 kindreds lacking biallelic AIRE variants in exons or flanking intronic regions; 15 had Puerto Rican ancestry. Through whole-genome sequencing, we identified a deep intronic AIRE variant (c.1504-818 G>A) cosegregating with the disease in all 17 patients. We developed a culture system of AIRE-expressing primary patient monocyte-derived dendritic cells and demonstrated that c.1504-818 G>A creates a cryptic splice site and activates inclusion of a 109-base pair frame-shifting pseudoexon. We also found low-level AIRE expression in patient-derived lymphoblastoid cell lines (LCLs) and confirmed pseudoexon inclusion in independent extrathymic AIRE-expressing cell lines. Through protein modeling and transcriptomic analyses of AIRE-transfected human embryonic kidney 293 and thymic epithelial cell 4D6 cells, we showed that this variant alters the carboxyl terminus of the AIRE protein, abrogating its function. Last, we developed an antisense oligonucleotide (ASO) that reversed pseudoexon inclusion and restored the normal AIRE transcript sequence in LCLs. Thus, our findings revealed c.1504-818 G>A as a founder APECED-causing AIRE variant in the Puerto Rican population and uncovered pseudoexon inclusion as an ASO-reversible genetic mechanism underlying APECED.

Evaluating AlphaFold for Clinical Pharmacology and Pharmacogenetics: A Case-Study of Huntingtin Variants Linked to Huntington's Disease.

To evaluate the artificial intelligence (AI)-guided AlphaFold algorithm for studying the binding interactions of human huntingtin and the aggregation of huntingtin peptides. Variants of huntingtin protein implicated in Huntington's disease were used as a model system to evaluate AlphaFold. Variants of huntingtin and huntingtin peptides with polyglutamine tracts (PQT) containing 21, 31, 51, or 78 glutamines were studied. The 3-dimensional structures of huntingtin variants and their interactions with huntingtin-associated protein-40 (HAP40) were obtained. Aggregation experiments were conducted with peptide sequences corresponding to variants of PQT, amino terminal sequence (NTS) plus PQT, NTS plus PQT plus proline rich region (PRR), and the 300 amino acid sequence from the NTS through HEAT3 of huntingtin. Oligomerization experiments with 1, 3, 6, or 12 peptide sequences were used to assess the quaternary structures of aggregates. The PQT and PQT plus NTS peptides formed a helical secondary structure that formed a central core in the quaternary structure of the aggregates The PRR formed an extended type II polyproline helix that did not participate in central core the aggregates. The distance between the amino and carboxyl termini of disease-linked 31Q, 51Q, and 78Q variants of full-length huntingtin was prominently decreased compared to the 21Q huntingtin. The interaction of HAP40 with the 78Q variant increased the distance between the amino and carboxyl termini. AlphaFold identified key tertiary structure changes in human huntingtin that have been independently corroborated in experimental models. The results highlight the utility of AlphaFold for hypothesis generation in pharmaceutical research.

An optimised method to isotopically label pure synthetic peptides 'in-house' for absolute quantification in bottom-up proteomics.

Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times. We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors. Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%. The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies.

Carboxy-terminal blockade of sortilin binding enhances progranulin gene therapy, a potential treatment for frontotemporal dementia.

The effectiveness of progranulin gene therapy in models of FTD is improved by blocking the protein's carboxy terminus, which prevents sortilin binding.

Multi-site esterification: a tunable, reversible strategy to tailor therapeutic peptides for delivery.

Peptides are naturally potent and selective therapeutics with massive potential; however, low cell membrane permeability limits their clinical implementation, particularly for hydrophilic, anionic peptides with intracellular targets. To overcome this limitation, esterification of anionic carboxylic acids on therapeutic peptides can simultaneously increase hydrophobicity and net charge to facilitate cell internalization, whereafter installed esters can be cleaved hydrolytically to restore activity. To date, however, most esterified therapeutics contain either a single esterification site or multiple esters randomly incorporated on multiple sites. This investigation provides molecular engineering insight into how the number and position of esters installed onto the therapeutic peptide α carboxyl terminus 11 (αCT11, RPRPDDLEI) with 4 esterification sites affect hydrophobicity and the hydrolysis process that reverts the peptide to its original form. After installing methyl esters onto αCT11 using Fischer esterification, we isolated 5 distinct products and used 2D nuclear magnetic resonance spectroscopy, reverse-phase high performance liquid chromatography, and mass spectrometry to determine which residues were esterified in each and the resulting increase in hydrophobicity. We found esterifying the C-terminal isoleucine to impart the largest increase in hydrophobicity. Monitoring ester hydrolysis showed the C-terminal isoleucine ester to be the most hydrolytically stable, followed by the glutamic acid, whereas esters on aspartic acids hydrolyze rapidly. LC-MS revealed the formation of transient intramolecular aspartimides prior to hydrolysis to carboxylic acids. In vitro proof-of-concept experiments showed esterifying αCT11 to increase cell migration into a scratch, highlighting the potential of multi-site esterification as a tunable, reversible strategy to enable the delivery of therapeutic peptides.

Comprehensive posttranslational modifications in the testis-specific histone variant H3t protein validated in tagged knock-in mice

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo.

Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20–30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.

Abstract Mo061: The major voltage-gated sodium channel accessory subunit in working ventricular myocytes may be β1B, not β1

Voltage-gated sodium channels, crucial for the action potential upstroke in cardiomyocytes, consist primarily of a single pore-forming α-subunit and 1 or 2 accessory β-subunits. SCN1B encodes two variants: β1, a transmembrane cell adhesion molecule, and β1B, typically described as a soluble secreted protein. While β1 and β1B share identical sequences in the amino-terminal (NT) extracellular Ig domain, they differ in their carboxy-terminal (CT) sequence. The Ig domain of β1/β1B has been deemed critical in maintaining normal cardiac conduction by various groups. Traditionally, effects on cardiac conduction have been attributed to β1, despite difficulties in detecting it at the protein level in the heart. We hypothesized that β1B, rather than β1, plays a significant role in electrical activity propagation in the heart. Utilizing an NT antibody (detects both variants), a β1-specific CT antibody, and a custom β1B-specific CT antibody, we examined SCN1B gene product profiles in various cardiac cell types, including neonatal rat ventricular myocytes and fibroblasts (NRVMs and NFs), isolated adult mouse cardiomyocytes (MCMs), and tissue slices from isolated adult rat hearts. The β1-specific CT antibody failed to label any bands in Western Blots (WB) of these cells and tissues, whereas the NT antibody detected a 37 kD band in all cell types and showed high levels of localization at cell-cell contacts using immunofluorescence (IF) with NRVMs. Conversely, the CT antibody showed minimal immunolabeling evidence. Moreover, an inhibitor of β1 Regulated Intramembrane Proteolysis failed to generate the expected 19 kD fragment if β1 were present in NRVMs. In adult rat hearts, the NT antibody labeled intercalated discs, while the CT showed minimal signal. Conversely, in WBs of NRVMs using our custom β1B antibody, we observed a 37 kD band consistent with the presence of β1B, and in heart sections, we found labeling of intercalated discs. These findings suggest that the major β subunit in working ventricular myocytes may be β1B, not β1. Further investigation is warranted, as these results could significantly impact our understanding of the roles and functional effects of β1 and β1B on cardiac conduction.

Abstract Tu005: Small Extracellular Vesicles Derived from Bovine Milk Contain an Endogenous Carboxyl Terminal Polypeptide of The Gap Junction Protein Connexin-43

Background: Connexin 43 (Cx43) is the body's most widely expressed gap junctional connexin. Researchers have shown evidence of non-canonical functions of Cx43 and the therapeutic promise of Cx43 mimetic peptides in cardiac pathologies. However, some critical barriers, including in vivo stability and potential immunotoxicity of Cx43 mimetic peptides, hinder clinical translation of these pro-drugs. To address these challenges, we are pioneering bovine milk-derived small extracellular vesicles (mEVs) as delivery vehicles for peptidic drugs. While exploring mEVs as drug delivery tools for αCT1 – a peptide mimicking Cx43 carboxyl terminus (CT) undergoing Phase III testing and known for promoting wound healing and cardioprotection - mEVs without loaded drug cargo exhibited similar bioactivity. This study aimed to test the hypothesis that Cx43, particularly CT fragments of Cx43, are naturally present in mEVs. Methods: mEVs were isolated from bovine milk using a novel protocol developed by our lab based on Tangential Flow Filtration and characterized by Western Blotting (WB), Nanoparticle Tracking Analysis, and Transmission Electron Microscopy. To determine the presence of Cx43 in mEVs, we used WB, immunoprecipitation and Single-Molecule Localization Microscopy (SMLM). Results: Characterization of mEVs confirmed relatively pure and ultrastructurally definitive mEVs isolated from milk at high density. WB for Cx43 in mEVs lysates based on two antibodies - one against the cytoplasmic loop domain and the other against CT-most 20 amino acids of Cx43, showed that full-length Cx43 was present in mEVs. Interestingly, the Cx43 CT antibody also detected a band at around 16 KDa, and this was confirmed by WB by using two more Cx43 CT antibodies. We also immunoprecipitated this Cx43 CT polypeptide from mEVs lysates using the CT antibodies. SMLM suggested that a subpopulation of mEVs contain Cx43 and demonstrated vesicle-to-vesicle heterogeneity to Cx43 immunolabeling. Conclusions: We show for the first time that an endogenous Cx43 CT polypeptide is present in mEVs. Our results provide new insight into the role of Cx43 in mEV bioactivity and potentially offer a path to safe, effective, and orally available therapeutic options based on mEVs.