Promiscuous Biotin Ligase BirA Research Articles

Proteomics of Protein Trafficking

Organs are interdependent and specialized – they communicate to enable organismal processes including growth, energy metabolism, and reproduction. Secreted peptides and proteins are essential regulators of homeostasis, and they compose a poorly-characterized communication network. Secreted factors are also relevant for disease: obesity affects 40% of the US population and results from poorly-characterized dysfunctional systemic signaling. Despite their relevance to homeostasis and disease, many additional factors remain to be identified. Characterizing the factors involved has been challenging, because conventional methods cannot identify low-abundance proteins or their origins and destinations. The broad goal of my new laboratory at Scripps is to apply novel high-throughput quantitative approaches we developed to interorgan communication in mammals, in order to understand the identity of the factors involved, which organs they originate from and target, their functions, and regulation of their production in disease. In this ASBMB Annual Meeting talk, I will describe our recent work and progress in establishing a proteomic platform, in which we biotinylate all proteins in a subcellular compartment of one organ using the engineered promiscuous biotin ligase BirA*G3 (a relative of TurboID), and then affinity-enrich the biotinylated proteins, identify them from distant organs using quantitative proteomics. This platform therefore allows simplifying the proteome and identifying low-abundance proteins, as well as their organs of origin and destination. Using this system in Drosophila, we identified 269 fat body secreted proteins in muscles, which included growth factors, cytokines, and proteins with receptors in muscles. In particular, I will present the characterization of muscle phenotypes of a fat body-derived protein CG2145. In addition to these data, I will describe our ongoing work on transitioning this method to mammalian systems. Altogether, this approach should be widely applicable to studies of secretome trafficking networks in vivo, including non-conventional secretion, in both arthropod and mammalian systems, in healthy or diseased states.

A proximity-labeling proteomic approach to investigate invadopodia molecular landscape in breast cancer cells

Metastatic progression is the leading cause of mortality in breast cancer. Invasive tumor cells develop invadopodia to travel through basement membranes and the interstitial matrix. Substantial efforts have been made to characterize invadopodia molecular composition. However, their full molecular identity is still missing due to the difficulty in isolating them. To fill this gap, we developed a non-hypothesis driven proteomic approach based on the BioID proximity biotinylation technology, using the invadopodia-specific protein Tks5α fused to the promiscuous biotin ligase BirA* as bait. In invasive breast cancer cells, Tks5α fusion concentrated to invadopodia and selectively biotinylated invadopodia components, in contrast to a fusion which lacked the membrane-targeting PX domain (Tks5β). Biotinylated proteins were isolated by affinity capture and identified by mass spectrometry. We identified known invadopodia components, revealing the pertinence of our strategy. Furthermore, we observed that Tks5 newly identified close neighbors belonged to a biologically relevant network centered on actin cytoskeleton organization. Analysis of Tks5β interactome demonstrated that some partners bound Tks5 before its recruitment to invadopodia. Thus, the present strategy allowed us to identify novel Tks5 partners that were not identified by traditional approaches and could help get a more comprehensive picture of invadopodia molecular landscape.

Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID.

Biotin proximity labeling has largely extended the toolbox of mass spectrometry-based interactomics. To date, BirA, engineered BirA variants, or other biotinylating enzymes have been widely applied to characterize protein interactions. By implementing chromatin purification-based methods the genome-wide interactome of proteins can be defined. However, acquiring a high-resolution interactome of a single genomic locus preferably by multiplexed measurements of several distinct genomic loci in parallel remains challenging. We recently developed CasID, a novel approach where the catalytically inactive Cas9 (dCas9) is coupled to the promiscuous biotin ligase BirA (BirA∗). With CasID, first the local proteome at repetitive telomeric, major satellite, and minor satellite regions was determined. With more efficient biotin ligases and sensitive mass spectrometry, others have successfully identified the chromatin composition at even smaller genomic, non-repetitive regions of a few hundred base pairs in length. Here, we summarize the most recent developments towards interactomics at a single genomic locus and provide a step-by-step protocol based on the CasID approach.

Using BioID for the Identification of Interacting and Proximal Proteins in Subcellular Compartments in Toxoplasma gondii.

BioID is an in vivo biotinylation system developed to examine the proximal and interacting proteins of a bait protein within a subcellular compartment. This approach has been exploited in Toxoplasma for protein-protein interaction studies and proteomic characterizations of intracellular compartments. The BioID method requires constructing a translational fusion between a protein of interest and the promiscuous biotin ligase BirA∗ (a mutant of the E. coli protein BirA) which enables trafficking of the protein to the correct intracellular compartment and association with its partners. Proximity labelling occurs upon addition of biotin to the media and the biotinylated target proteins are then be purified using stringent conditions via streptavidin chromatography. In this chapter, we describe the methodology to fuse BirA∗(or the newer variant BioID2) to a bait protein using endogenous gene tagging in Toxoplasma and then identify the proximal and interacting proteins using in vivo biotinylation, streptavidin purification and mass spectrometric analysis.

Stromal Interaction Molecule and Gelsolin Mediate Pathological Cardiac Remodeling

Pathological cardiac remodeling is characterized by a series of poorly understood changes in myocardial microstructure involving changes in the cardiomyocytes cytoskeleton. Stromal interaction molecule 1 (STIM1) resides mostly in the sarco/endoplasmic reticulum where is senses intraluminal Ca2+ levels and activates the Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 leading to cytoskeletal remodeling are not known. Proximity‐labeling technique (BioID) can capture weak and transient protein‐protein interactions. Hence, in the present study we investigated the STIM1 interactome used the BioID technique. A promiscuous biotin ligase BirA* was fused to the cytoplasmic C‐terminous of STIM1 and generated MEF stable cell line expressing STIM1‐BirA*. Biotin was added to the culture medium of MEF expressing STIM1‐BirA as well as in control cells not expressing STIM1‐BirA. Cells were harvested after twenty‐four hours and total lysates were incubated with streptavidin magnetic beads to pull down the biotinylated proteins which were subsequently identified by mass spectrometry. STIM1 interactors were validated in neonatal rat cardiac myocytes (NRCM) coupled with biochemical, immuno‐staining techniques and confocal microscopy. Screening of biotinylated proteins identified several high confidence known and previously unknown interactors of STIM1. One of the novel interactors was Gelsolin (GNS), a Ca2+‐dependent actin‐severing protein that is a key regulator of actin filament assembly and disassembly. STIM1 and GSN relationship was further investigated using NRCM. We show that STIM1 upregulation in NRCM was enough to induce upregulation of GNS protein levels. Immunostaining confirms colocalization between STIM1 and GSM. Furthermore, STIM1 overexpression in NVCM show altered actin dynamic and tubulin overexpression probably in response to cytoskeletal instability. We report the first use of BioID to characterize the STIM1 interactome. Our work unravels an unprecedented role of GSN as a part of the STIM1 interactome and its role in the modulation of the actin cytoskeleton.Support or Funding InformationWe gratefully acknowledge funding from the the National Heart, Lung and Blood Institute (award HL114869, to Dr. S.M.), and from UTHSC seed funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defining the Role of Protein Interactions at WRAMP Structures in Directional Migration

Cell migration is a critical process in development, tissue regeneration, and cancer cell invasion, but mechanisms that establish the location of the cell rear and activate rear contractile forces are still poorly understood. Our lab previously discovered a protein network named the “Wnt5a‐receptor‐actomyosin‐polarity (WRAMP) structure” which may serve as a novel mechanism for rear‐directed control of migration. The WRAMP structure forms transiently in response to the signaling ligand Wnt5a and is characterized by polarization of F‐actin, myosin‐IIB, and melanoma cell adhesion molecule (MCAM). Cells with WRAMP structures migrate in a persistent direction for longer periods of time compared to cells without WRAMP structures, and the location where WRAMP structures form may determine the position of the cell rear and direction of migration. However, the specific interactions of proteins at WRAMP structures that activate actomyosin contraction are unknown. To address this question, we have generated plasmids to express previously identified WRAMP structure members MCAM and moesin fused to the engineered peroxidase APEX2 or the promiscuous biotin ligase BirA(R118G) for use in the proteomics techniques APEX and BioID, respectively. In these methods, proteins in close proximity to the bait protein are biotinylated and subsequently isolated on streptavidin beads for identification by mass spectrometry. Ongoing goals of this project are to compare APEX and BioID methods in melanoma cells to build up a WRAMP structure “interactome,” prioritized by interactions that increase in the presence of Wnt5a treatment. In addition, we are examining the role of RhoA/Rho kinase (ROCK) signaling in activation of actomyosin contraction using immunofluorescence for activated substrates of ROCK at WRAMP structures, and we will test a literature‐based model for RhoA activation through MCAM‐moesin interactions using an MCAM mutant which lacks moesin binding. These findings will help define the spatial organization of WRAMP structures and identify key interactions and signaling pathways involved in control of cell migration.Support or Funding InformationThis work is supported by funding from the National Institutes of Health (R01GM105997 and T32GM008759).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BioID Reveals Novel Proteins of the Plasmodium Parasitophorous Vacuole Membrane.

During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P.berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P.berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.

Determination of local chromatin composition by CasID

ABSTRACTChromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA. With super-resolution microscopy, we confirmed the localization of the putative transcription factor ZNF512 at chromocenters. The versatility of CasID facilitates the systematic elucidation of functional protein complexes and locus-specific chromatin composition.

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

ABSTRACTToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.

Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.