Arginylation of ⍺-tubulin at E77 regulates microtubule dynamics via MAP1S.

Arginylation is the posttranslational addition of arginine to a protein by arginyltransferase-1 (ATE1). Previous studies have found that ATE1 targets multiple cytoskeletal proteins, and Ate1 deletion causes cytoskeletal defects, including reduced cell motility and adhesion. Some of these defects have been linked to actin arginylation, but the role of other arginylated cytoskeletal proteins has not been studied.

β-actin function in platelets and red blood cells can be performed by γ-actin and is therefore independent of actin isoform protein sequence.

Actin is an essential component of the cytoskeleton in every eukaryotic cell. Cytoplasmic β-and γ-actin are over 99% identical to each other at the protein level, but are encoded by different genes and play distinct roles in vivo. Blood cells, especially red blood cells (RBC), contain almost exclusively β-actin, and it has been generally assumed that this bias is dictated by unique suitability of β-actin for RBC cytoskeleton function due to its specific amino acid sequence.

Alginate-Poly[2-(methacryloyloxy)ethyl]trimethylammonium Chloride (PMETAC) Immunoisolating Capsules Prolong the Viability of Pancreatic Islets In Vivo.

Background/objectives: This study focuses on the development and evaluation of novel alginate-poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMETAC) microcapsules for encapsulating pancreatic islets to address insulin deficiency in diabetes.

Methods: In previous research, we fabricated and characterized PMETAC microcapsules, evaluating their stability and permeability in vitro. This study further probes the capsules in vivo, focusing on the functional activity of the encapsulated islets post-transplantation, their viability extension, and the assessment of the immunoprotective, antifibrotic properties, and biostability of the capsules.

Amino acid-level differences in alpha tubulin sequences are uniquely required for meiosis.

Tubulin is the major structural constituent of the microtubule cytoskeleton. Yeast Schizosaccharomyces pombe contain two α- tubulins genes, nda2 and atb2, that are highly functionally distinct: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. The functional determinants underlying this distinction are unknown.

A Fresh Look at Islet Isolation from Rabbit Pancreases.

Islet transplantation represents a promising therapeutic approach for diabetes management, yet the isolation and evaluation of pancreatic islets remain challenging. This study focuses on the isolation of islets from rabbit pancreases, followed by a comprehensive assessment of their viability and functionality. We developed a novel method for isolating islet cells from the pancreas of adult rabbits.

Probing of New Polymer-Based Microcapsules for Islet Cell Immunoisolation.

Islet allotransplantation offers a promising cell therapy for type 1 diabetes, but challenges such as limited donor availability and immunosuppression persist. Microencapsulation of islets in polymer-coated alginate microcapsules is a favored strategy for immune protection and maintaining islet viability. This study introduces Poly [2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMETAC) as an innovative coating material for microcapsules.

Prospective fecal microbiomic biomarkers for chronic wasting disease.

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the United States. Here, we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the USA compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer.

The Effect of Diabetes Mellitus Type 1 on the Energy Metabolism of Hepatocytes: Multiphoton Microscopy and Fluorescence Lifetime Imaging.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level.

Global Analysis of Post-Translational Side-Chain Arginylation Using Pan-Arginylation Antibodies.

Arginylation is a post-translational modification mediated by the arginyltransferase 1 (ATE1), which transfers the amino acid arginine to a protein or peptide substrate from a tRNA molecule. Initially, arginylation was thought to occur only on N-terminally exposed acidic residues, and its function was thought to be limited to targeting proteins for degradation. However, more recent data have shown that ATE1 can arginylate side chains of internal acidic residues in a protein without necessarily affecting metabolic stability.

The N-degron pathway mediates lipophagy: The chemical modulation of lipophagy in obesity and NAFLD.

Background And Aims: Central to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) is the accumulation of lipids in the liver and various fat tissues. We aimed to elucidate the mechanisms by which lipid droplets (LDs) in the liver and adipocytes are degraded by the autophagy-lysosome system and develop therapeutic means to modulate lipophagy, i.e.

Poly(3-hydroxybutyrate) 3D-Scaffold-Conduit for Guided Tissue Sprouting.

Scaffold biocompatibility remains an urgent problem in tissue engineering. An especially interesting problem is guided cell intergrowth and tissue sprouting using a porous scaffold with a special design. Two types of structures were obtained from poly(3-hydroxybutyrate) (PHB) using a salt leaching technique.

-actin is essential for structural integrity and physiological function of the retina.

Lack of non-muscle -actin gene (Actb) leads to early embryonic lethality in mice, however mice with - to -actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated de-generation of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS.

Development of New Tools for the Studies of Protein Arginylation.

Studies of posttranslational modifications present many unique challenges, stemming from their role as the major drivers of biological complexity. Perhaps the most immediate challenge to researchers working on virtually any posttranslational modification is the shortage of reliable easy-to-use tools that can enable massive identification and characterization of posttranslationally modified proteins, as well as their functional modulation in vitro and in vivo. In the case of protein arginylation, which utilizes charged Arg-tRNA that is also used by the ribosomes, detection and labeling of arginylated proteins is especially difficult, because of the necessity of distinguishing these proteins from the products of conventional translation.

Analysis of Arginylated Peptides by Subtractive Edman Degradation.

During the early studies of N-terminal arginylation, Edman degradation was widely used to identify N-terminally added Arg on protein substrates. This old method is reliable, but highly depends on the purity and abundance of samples and can become misleading unless a highly purified highly arginylated protein can be obtained. Here, we report a mass spectrometry-based method that utilizes Edman degradation chemistry to identify arginylation in more complex and less abundant protein samples.

Identification of Arginylated Proteins by Mass Spectrometry.

Here, we describe the method for the identification of arginylated proteins by mass spectrometry. This method has been originally applied to the identification of N-terminally added Arg on proteins and peptides and then expanded to the side chain modification which has been recently described by our groups. The key steps in this method include the use of the mass spectrometry instruments that can identify peptides with very high pass accuracy (Orbitrap) and apply stringent mass cutoffs during automated data analysis, followed by manual validation of the identified spectra.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

High-Throughput Arginylation Assay in Microplate Format.

Here, we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for the identification of small molecule inhibitors and activators of ATE1, high-volume analysis of AE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified 2 compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.

Assaying ATE1 Activity In Vitro.

Here, we describe a standard arginyltransferase assay in vitro using bacterially expressed purified ATE1 in a system with a minimal number of components (Arg, tRNA, Arg-tRNA synthetase, and arginylation substrate). Assays of this type have first been developed in the 1980s using crude ATE1 preparations from cells and tissues and then perfected recently for the use with bacterially expressed recombinant protein. This assay represents a simple and efficient way to measure ATE1 activity.

Enzymatic Aminoacylation of tRNA Using Recombinant Arg-tRNA Synthetase.

This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g.

Preparation of an Enriched tRNA Fraction for Arginylation by Expression in E. coli.

The method described here provides a fast and efficient way to obtain an enriched preparation of tRNA of interest, which is also posttranscriptionally modified by the intracellular machinery of the host cells, E. coli. While this preparation also contains a mixture of total E.

Preparation of tRNA for Arginylation Assay by In Vitro Transcription.

This chapter describes the preparation of tRNA by in vitro transcription. tRNA produced by this method can be efficiently utilized for in vitro arginylation assays, following aminoacylation with Arg-tRNA synthetase, either directly during the arginylation reaction or separately to produce the purified preparation of Arg-tRNA. tRNA charging is described in other chapters of this book.

Bacterial Expression and Purification of Recombinant Arginyltransferase (ATE1) and Arg-tRNA Synthetase (RRS) for Arginylation Assays.

Here, we describe the procedure for the expression and purification of recombinant ATE1 from E. coli. This method is easy and convenient and can result in one-step isolation of milligram amounts of soluble enzymatically active ATE1 at nearly 99% purity.

Assaying Intracellular Arginylation Activity Using a Fluorescent Reporter.

In this chapter, we present a simplified version of the method described in Chapter 9 of this book, adapted for fast and convenient evaluation of intracellular arginylation activity in live cells. As in the previous chapter, this method utilizes a GFP-tagged N-terminal β-actin peptide transfected into cells as a reporter construct. Arginylation activity can then be evaluated by harvesting the reporter-expressing cells and analyzing them directly by Western blot using an arginylated β-actin antibody and a GFP antibody as an internal reference.

Assaying ATE1 Activity in Yeast by β-Gal Degradation.

In the 1980s, it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates.