APEX2

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation...

The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied...

Fluorescent tagging of biomolecules enables their sensitive detection during separation and determining their subcellular location. In this context, peroxidase-based reactions are actively utilized for signal amplification. To harness this potential, we developed a genetically encodable enzymatic fluorescence signal amplification method using APEX (FLEX). We synthesized a fluorescent probe, Jenfluor triazole (JFT1), which effectively amplifies and restricts fluorescence signals under fixed conditions, enabling fluorescence-based detection of subcellularly localized electron-rich metabolites...

There is an urgent requirement to acquire a comprehensive comprehension of novel therapeutic targets for prostate cancer to facilitate the development of medications with innovative mechanisms. In this study, we identified gambogic acid (GBA) as a specific pyroptosis inducer in prostatic cancer cells. By using a thermal proteome profiling (TPP) strategy, we revealed that GBA induces pyroptosis by directly targeting the canopy FGF signaling regulator (CNPY3), which was previously considered "undruggable". Moreover, through the utilization of the APEX2-based proximity labeling method, we found that GBA recruited delactatease SIRT1, resulting in the elimination of lysine lactylation (Kla) on CNPY3...

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion...

UNLABELLED: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor...

It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac...

KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to ∼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions...

GGGGCC hexanucleotide repeat expansion in C9orf72 causes frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Expanded GGGGCC repeat RNA accumulates within RNA foci and is translated into toxic dipeptide repeat proteins; thus, efficient repeat RNA degradation may alleviate diseases. hnRNPA3, one of the repeat RNA-binding proteins, has been implicated in the destabilization of repeat RNA. Using APEX2-mediated proximity biotinylation, here, we demonstrate PABPC1, a cytoplasmic poly (A)-binding protein, interacts with hnRNPA3...

Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins...

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO)...

Currently available genetically encoded H2 O2 probes report on the thiol redox state of the probe, which means that they reflect the balance between probe thiol oxidation and reduction. Here we introduce the use of the engineered heme peroxidase APEX2 as a thiol-independent chemogenetic H2 O2 probe that directly and irreversibly converts H2 O2 molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, specificity, and the ability to quantitate endogenous H2 O2 turnover. We show how the probe can be used to detect changes in endogenous H2 O2 generation and to assess the roles and relative contributions of endogenous H2 O2 scavengers...

In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration...

Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues...

Unraveling interacting partners of protein tyrosine (Tyr) phosphatases is considered a key aspect in resolving the regulation of signaling cascades either in a pathological or in developmental context. Mass spectrometry (MS)-based protein identification has emerged as the major approach in this arena, complemented by the development of novel biochemical methodologies for sample preparation. In this chapter, we highlight two methods that, combined with mass spectrometry, may help the investigator create an interactome map for the phosphatase of interest within a specific biological context...

BACKGROUND: Ovarian cancer (OC) is a highly heterogeneous and malignant gynecological cancer, thereby leading to poor clinical outcomes. The study aims to identify and characterize clinically relevant subtypes in OC and develop a diagnostic model that can precisely stratify OC patients, providing more diagnostic clues for OC patients to access focused therapeutic and preventative strategies. METHODS: Gene expression datasets of OC were retrieved from TCGA and GEO databases...

Adipocyte lipid droplets (LDs) play a crucial role in systemic lipid metabolism by storing and releasing lipids to meet the organism's energy needs. Hormonal signals such as catecholamines and insulin act on adipocyte LDs, and impaired responsiveness to these signals can lead to uncontrolled lipolysis, lipotoxicity, and metabolic disease. To investigate the mechanisms that control LD function in human adipocytes, we applied proximity labeling mediated by enhanced ascorbate peroxidase (APEX2) to identify the interactome of PLIN1 in adipocytes differentiated from human mesenchymal progenitor cells...

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis...

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) has the highest mortality rate among malignant tumors worldwide. This study aimed to analyze the biological characteristics of serum proteins in hepatitis B (HBV)-related liver diseases, identify diagnostic biomarkers for HBV-infected HCC, and provide a scientific basis for its prevention and treatment. MATERIALS AND METHODS: We used HuProt arrays to identify candidate biomarkers for HBV-related liver diseases and verified the differential biomarkers by using an HCC-focused array...

Although APEX2-mediated proximity labeling has been extensively implemented for studying RNA subcellular localization in live cells, the biotin-phenoxyl radical used for labeling RNAs has a relatively low efficiency, which can limit its compatibility with other profiling methods. Herein, a set of phenol derivatives were designed as APEX2 probes through balancing reactivity, hydrophilicity, and lipophilicity. Among these derivatives, Ph_N3 exhibited reliable labeling ability and enabled two biotinylation routes for downstream analysis...