We designed a platform for monitoring the degradation of exogenous proteins in live cells. We engineered a semi-synthetic platform, which consists of Enhanced Green Fluorescent Protein tagged with SpyCatcher to enable its conjugation to a SpyTag peptide bearing a Von Hippel-Lindau E3 ligand, which was delivered to live cells to promote its degradation. This platform lays the ground for studying the degradation of endogenous proteins equipped with SpyTag and for tracking the degradation of post-translationally modified proteins in live cells.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1002/cbic.202300731 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD).
J Vis Exp
January 2025
Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Henry and Allison McCance Center for Brain Health, Department of Neurology, Massachusetts General Hospital, Harvard Medical School;
A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of Alzheimer's disease (AD) is presented here. To begin, fresh human neuro progenitor ReN cells expressing β-amyloid precursor protein (APP) containing familial Alzheimer's disease (FAD) or naïve ReN cells are grown in thin (1:100) Matrigel-coated tissue culture plates. After the cells reach confluency, these are electroporated with expression plasmids encoding red fluorescence protein (RFP)-conjugated mitochondria-binding sequence of AKAP1(34-63) (Mito-RFP) that detects mitochondria or constitutive MAM stabilizers MAM 1X or MAM 9X that stabilize tight (6 nm ± 1 nm gap width) or loose (24 nm ± 3 nm gap width) MAMs, respectively.
View Article and Find Full Text PDFEnvironmental desiccation stress induces viable but non culturable state in Neisseria meningitidis.
Arch Microbiol
January 2025
Symbiosis School of Biological Sciences, Symbiosis International (Deemed University)Lavale, Pune, Maharashtra, 412115, India.
Environmental factors play a crucial role in bacterial virulence. During transmission, in a non-host environment bacteria are exposed to various environmental stress which could alter bacterial physiology and virulence. N.
View Article and Find Full Text PDFQuantitative Profiling pH Heterogeneity of Acidic Endolysosomal Compartments using Fluorescence Lifetime Imaging Microscopy.
Mol Biol Cell
January 2025
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147 USA.
The endo-lysosomal system plays a crucial role in maintaining cellular homeostasis and promoting organism fitness. The pH of its acidic compartments is a crucial parameter for proper function, and it is dynamically influenced by both intracellular and environmental factors. Here, we present a method based on fluorescence lifetime imaging microscopy (FLIM) for quantitatively analyzing the pH profiles of acidic endolysosomal compartments in diverse types of primary mammalian cells and in live organism .
View Article and Find Full Text PDFMitochondria-plasma membrane contact sites regulate the ER-mitochondria encounter structure.
J Cell Sci
January 2025
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
Cells form multiple, molecularly distinct membrane contact sites (MCSs) between organelles. Despite knowing the molecular identity of several of these complexes, little is known about how MCSs are coordinately regulated in space and time to promote organelle function. Here, we examined two well-characterized mitochondria-ER MCSs - the ER-Mitochondria encounter structure (ERMES) and the mitochondria-ER-cortex anchor (MECA).
View Article and Find Full Text PDFPEBP1 amplifies mitochondrial dysfunction-induced integrated stress response.
Elife
January 2025
Centre for Cellular Biology and Signalling, Zhejiang University-University of Edinburgh (ZJU-UoE) Institute, Haining, China.
Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing mass spectrometry-based cellular thermal shift assay (MS-CETSA), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!