Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed ...unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins—structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Display omitted
•Global calcineurin signaling in humans revealed through systematic substrate mapping•Discovery of calcineurin-binding sequences enables robust in silico SLiM predictions•BioID uncovers SLiM-dependent calcineurin proximity to nuclear pores and centrosomes•Calcineurin dephosphorylates nuclear pore proteins and regulates transport in vivo
Wigington, Roy, et al. elucidate a signaling network for the Ca2+-activated phosphatase calcineurin by systematically uncovering calcineurin-binding SLiMs throughout the human proteome. Using in vitro, in silico, and in vivo methods, they discover multiple calcineurin substrates, including Notch1 and several nucleoporins, leading to the unexpected finding that calcineurin regulates nuclear transport.
Proximity-dependent biotinylation approaches such as BioID and APEX overcome classical limitations of biochemical purification and have gained widespread use in recent years for revealing cellular ...neighborhoods. Here we focus on the structural diversity and mechanisms of the two classes of enzymes, biotin protein ligases and peroxidases, and discuss current and emerging applications of these enzymes for proximity dependent biotinylation. We provide guidelines for enzyme selection and experimental design for performing and interpreting proximity-dependent biotinylation experiments.
Display omitted
Highlights
•Proximity-dependent biotinylation (PDB) approaches involve fusion of a bait with an enzyme.•BioID (biotin protein ligase) and APEX (peroxidase) are distinct enzymes used in PDB.•Past, present and future development and applications of PDB are discussed.•We review labeling mechanisms and kinetics to provide guidance for experimental design.•We discuss controls and considerations for data interpretation.
The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.
In the last 10 years, proximity‐dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from specific protein complexes to ...entire compartments. This is achieved by using enzymes such as BirA* and APEX that are fused to proteins of interest and biotinylate proteins in their proximity. PDB techniques are now also increasingly used in apicomplexan parasites. In this review, we first give an overview of the main PDB approaches and how they compare with other techniques that address similar questions. PDB is particularly valuable to detect weak or transient protein associations under physiological conditions and to study cellular structures that are difficult to purify or have a poorly understood protein composition. We also highlight new developments such as novel smaller or faster‐acting enzyme variants and conditional PDB approaches, providing improvements in both temporal and spatial resolution which may offer broader application possibilities useful in apicomplexan research. In the second part, we review work using PDB techniques in apicomplexan parasites and how this expanded our knowledge about these medically important parasites.
Proximity‐dependent biotinylation (PDB) approaches using PDB enzymes such as BirA* and APEX have emerged as powerful tools for interactome and compartment proteome studies. Here we describe the general properties, principles, and recent improvements of PDB. We summarize PDB‐derived insights into the biology of apicomplexan parasites and highlight the great promise of PDB for further discoveries in these parasites.
At present, it remains difficult to deconvolute serum in order to identify the cell or tissue origin of a given circulating protein. Here, by exploiting the properties of proximity biotinylation, we ...describe a mouse model that enables the elucidation of the in vivo tissue-specific secretome. As an example, we demonstrate how we can readily identify in vivo endothelial-specific secretion as well as how this model allows for the characterization of muscle-derived serum proteins that either increase or decrease with exercise. This genetic platform should, therefore, be of wide utility in understanding normal and disease physiology and for the rational design of tissue-specific disease biomarkers.
Mycoplasma bovis ( M. bovis ) is a significant bovine pathogen associated with various diseases, including bovine bronchopneumonia and mastitis resulting in substantial economic losses within the ...livestock industry. However, the development of effective control measures for M. bovis is hindered by a limited understanding of its virulence factors and pathogenesis. Nucleomodulins are newly identified secreted proteins of bacteria that internalize the host nuclei to regulate host cell gene expression and serve as critical virulence factors. Although recent reports have initiated exploration of mycoplasma nucleomodulins, the efficiency of conventional techniques for identification is very limited. Therefore, this study aimed to establish high-throughput methods to identify novel nucleomodulins of M. bovis . Using a direct biotinylation (DB) approach, a total of 289 proteins were identified including 66 high abundant proteins. In parallel, the use of proximity-based biotinylation (PBB), identified 28 proteins. Finally, seven nucleomodulins were verified to be nuclear by transfecting the bovine macrophage cell line BoMac with the plasmids encoding EGFP-fused proteins and observed with Opera Phenix, including the known nucleomodulin MbovP475 and six novel nucleomodulins. The novel nucleomodulins were four ribosomal proteins (MbovP599, MbovP678, MbovP710, and MbovP712), one transposase (MbovP790), and one conserved hypothetical protein (MbovP513). Among them, one unique nucleomodulin MbovP475 was identified with DB, two unique nucleomodulins (MbovP513 and MbovP710) with PBB, and four nucleomodulins by both. Overall, these findings established a foundation for further research on M. bovis nucleomodulin-host interactions for identification of new virulence factors.
The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized ...processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP's ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.
The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins ...that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the target antigen to be captured and identified by mass spectrometry. We demonstrated the specificity and sensitivity of our method by examining the well-studied mitochondrial matrix. We then used the method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. The ability to detect proximal proteins and putative interactors in intact tissues, and to quantify changes caused by different conditions or in the presence of disease mutations, can provide a window into cell biology and disease pathogenesis.
The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization ...has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
Persistent intranuclear infection is an uncommon infection strategy among RNA viruses. However, Borna disease virus 1 (BoDV‐1), a nonsegmented, negative‐strand RNA virus, maintains viral infection in ...the cell nucleus by forming structured aggregates of viral ribonucleoproteins (vRNPs), and by tethering these vRNPs onto the host chromosomes. To better understand the nuclear infection strategy of BoDV‐1, we determined the host protein interactors of the BoDV‐1 large (L) protein. By proximity‐dependent biotinylation, we identified several nuclear host proteins interacting with BoDV‐1 L, one of which is TRMT112, a partner of several methyltransferases (MTases). TRMT112 binds with BoDV‐1 L at the RNA‐dependent RNA polymerase domain, together with BUD23, an 18S ribosomal RNA MTase and 40S ribosomal maturation factor. We then discovered that BUD23–TRMT112 mediates the chromosomal tethering of BoDV‐1 vRNPs, and that the MTase activity is necessary in the tethering process. These findings provide us a better understanding on how nuclear host proteins assist the chromosomal tethering of BoDV‐1, as well as new prospects of host–viral interactions for intranuclear infection strategy of orthobornaviruses.
PDF
