BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates

The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF^β-TrCP1 and SCF^β-TrCP2 are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF^β-TrCP1/2 interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF^β-TrCP1/2 substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.More than 600 putative ubiquitin E3 ligases are encoded in the human genome (1, 2). Although a number of these proteins are known to play critical roles in human health (1–4), the specific biological functions—and substrates—of most E3s remain poorly characterized. The identification of E3 substrates has been difficult in part because: (1) ligase - substrate interactions are often of low affinity (generally in the high nm to microMolar (μM) range) and/or of a transient nature; (2) many substrates are subjected to rapid proteasomal degradation and are therefore not available for detection; (3) the human ubiquitome is extremely complex, and; (4) many substrate proteins are localized to poorly soluble cellular compartments, making their isolation and identification by standard immunoprecipitation (IP)-based techniques extremely challenging (1–4).Methods such as protein chip (5) and yeast two-hybrid screening (6) have been used to identify a limited number of E3-substrate interactions. However, these methods are not conducted in live mammalian cells, and may not be generally applicable for the identification of substrates of the hundreds of unique multi-protein E3 complexes (e.g. SCF, APC, VHL, etc.) or the identification of E3-substrate interactions that are dependent on specific types of post-translational modifications. Several putative inhibitors of apoptosis substrates were identified using the recently described NEDDylator technique (7), in which the E2 protein UBC12 (UBE2M) is fused to the E3 ligase of interest, allowing for the conjugation of the ubiquitin-like protein NEDD8 to substrates. Global protein stability profiling (8) and quantitative mass spectrometry methods (9) have also been used successfully to identify E3 targets. However, because of their cost and/or complexity, and the challenges posed by the extremely large size of the human ubiquitome, these methods have not been widely adopted to date.Proximity-based biotinylation, or BioID¹ (10), is a new method developed for the characterization of protein-protein interactions in living cells. Briefly, a protein of interest is fused in-frame with an E. coli biotin conjugating enzyme mutant (BirA R118G, or BirA*). The BirA* moiety can efficiently activate biotin, but exhibits a reduced affinity for the activated molecule (11); biotinoyl-AMP thus simply diffuses away from BirA* and reacts with nearby amine groups - including those present on lysine residues in neighboring polypeptides. Following cell lysis, biotinylated proteins can be affinity purified using streptavidin and identified using mass spectrometry (Fig. 1A). Since interactors are covalently modified with biotin, robust lysis conditions can be used to solubilize polypeptides localized to poorly soluble cellular compartments (10, 12–16). Moreover, since this method does not require that protein-protein interactions be maintained post-lysis, weak and/or transient interactors may also be identified. We reasoned that BioID may be exploited to capture ubiquitin E3 ligase substrates and tested this notion here.Open in a separate windowFig. 1.BioID can be used to identify ubiquitin E3 ligase substrates. A, An N-terminal tag consisting of the FLAG epitope and the mutant E. coli biotin conjugating protein BirA R118G (BirA*) was fused to the N terminus of the human F-box proteins β-TrCP1 and β-TrCP2. The BirA* protein converts biotin (black hexagon) to biotinoyl-AMP (yellow hexagon). The mutant BirA protein exhibits a reduced affinity for the activated biotin molecule; biotinoyl-AMP thus diffuses away and reacts with free amine groups on lysine residues in nearby polypeptides, including e.g. the bait protein itself, other SCF complex components (CUL1, SKP1), SCF substrates (S) and substrate binding partners (A, B). In the presence of the 26S proteasome inhibitor MG132, β-TrCP substrates are stabilized. Following cell lysis under stringent buffer conditions, biotinylated proteins are affinity purified using streptavidin coupled to Sepharose beads. Streptavidin-bound proteins are washed and subjected to trypsin proteolysis, and the liberated peptides are identified using tandem mass spectrometry. B, Expression of FLAGBirA*-β-TrCP1/2 leads to biotinylation of endogenous proteins. 293 T-REx cells expressing FLAGBirA*-β-TrCP1 or FLAGBirA*-β-TrCP2 were treated with tetracycline (1 μg/ml) to induce protein expression, and with biotin (50 μm) to enable proximity-dependent polypeptide labeling. Whole cell lysates were subjected to SDS-PAGE and immunoblotted with an anti-FLAG antibody (top panel) or streptavidin-HRP (horseradish peroxidase; bottom panel). C, β-TrCP1/2 interactors displaying a substrate profile. Proteins identified in the BioID analysis with a ProteinProphet score ≥0.85 (corresponding to ≤1% FDR), a SAINT score ≥0.75, and a spectral count ratio (+MG132/untreated) log₂ >1. Circles, polypeptides identified in β-TrCP1 BioID; squares, proteins identified in β-TrCP2 BioID. Previously identified β-TrCP interactors are highlighted in blue. Proteins demonstrated in this study to be stabilized following β-TrCP1/2 knockdown (see Fig. 2 and Supplemental Fig. 2) are highlighted in green. D, Overlap of FLAG IP-MS and BioID substrate candidates. Diagram highlighting the overlap between BioID hits displaying a substrate profile (pink), FLAG IP-MS hits displaying a substrate profile (yellow), and previously reported β-TrCP interactors (blue). E, Functional categories of FLAG IP-MS and BioID substrate candidates. Numbers of previously reported β-TrCP interactors within each category are indicated in blue and numbers of new substrate candidates in each category indicated in red.The human beta transducin repeat-containing polypeptides β-TrCP1 (FBXW1) and β-TrCP2 (FBXW11) are evolutionarily conserved paralogous F-box proteins sharing >80% amino acid sequence identity and >90% homology, and act as substrate recognition components of SCF (Skp1-Cullin-F-box) complexes (17). A number of β-TrCP substrates have been well documented, implicating these ligases in numerous biological functions, including regulation of the NFκB, Hippo, Wnt and Hedgehog signaling pathways (18–21). Some β-TrCP targets harbor the sequence DSGX(n)S or variants thereof. Phosphorylation of this sequence is required for SCF^β-TrCP1/2 binding and thus regulates the half-life of these “phosphodegron”-containing proteins (21, 22). However, other bona fide SCF^β-TrCP1/2 substrates contain highly degenerate or non-canonical degrons, which are thought to mediate constitutive turnover (21). A single, simple linear sequence motif that could predict β-TrCP binding has thus not been defined.Here we demonstrate that BioID performed on cells treated with the proteasome inhibitor MG132 can recover many of the previously characterized substrates and stable interactors of β-TrCP1/2. Using semi-quantitative mass spectrometry, we identify and validate a number of new substrates, linking these well-studied E3 ligases to several new biological functions. The method used here is simple, scalable, and should be broadly applicable for the identification of substrates for many other E3s.     

Variability in the Practice of Fertility Preservation for Patients with Cancer

Fertility is important to women and men with cancer. While options for fertility preservation (FP) are available, knowledge regarding the medical application of FP is lacking. Therefore we examined FP practices for cancer patients among reproductive endocrinologists (REs). A 36 item survey was sent to board-certified REs. 98% of respondents reported counseling women with cancer about FP options. Oocyte and embryo cryopreservation were universally offered by these providers, but variability was noted in reported management of these cases—particularly for women with breast cancer. 86% of the respondents reported using letrozole during controlled ovarian stimulation (COS) in patients with estrogen receptor positive (ER+) breast cancer to minimize patient exposure to estrogen. 49% of respondents who reported using letrozole in COS for patients with ER+ breast cancer reported that they would also use letrozole in COS for women with ER negative breast cancer. Variability was also noted in the management of FP for men with cancer. 83% of participants reported counseling men about sperm banking with 22% recommending against banking for men previously exposed to chemotherapy. Overall, 79% of respondents reported knowledge of American Society for Clinical Oncology FP guidelines—knowledge that was associated with providers offering gonadal tissue cryopreservation (RR 1.82, 95% CI 1.14–2.90). These findings demonstrate that RE management of FP in cancer patients varies. Although some variability may be dictated by local resources, standardization of FP practices and communication with treating oncologists may help ensure consistent recommendations and outcomes for patients seeking FP.     

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.     

An actin-dependent conformational change in myosin

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.     

Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans

Mammalian nuclear hormone receptors (NHRs), such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs), precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid β-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.     

A Within-Animal Comparison of Skilled Forelimb Assessments in Rats

A variety of skilled reaching tasks have been developed to evaluate forelimb function in rodent models. The single pellet skilled reaching task and pasta matrix task have provided valuable insight into recovery of forelimb function in models of neurological injury and disease. Recently, several automated measures have been developed to reduce the cost and time burden of forelimb assessment in rodents. Here, we provide a within-subject comparison of three common forelimb assessments to allow direct evaluation of sensitivity and efficiency across tasks. Rats were trained to perform the single pellet skilled reaching task, the pasta matrix task, and the isometric pull task. Once proficient on all three tasks, rats received an ischemic lesion of motor cortex and striatum to impair use of the trained limb. On the second week post-lesion, all three tasks measured a significant deficit in forelimb function. Performance was well-correlated across tasks. By the sixth week post-lesion, only the isometric pull task measured a significant deficit in forelimb function, suggesting that this task is more sensitive to chronic impairments. The number of training days required to reach asymptotic performance was longer for the isometric pull task, but the total experimenter time required to collect and analyze data was substantially lower. These findings suggest that the isometric pull task represents an efficient, sensitive measure of forelimb function to facilitate preclinical evaluation in models of neurological injury and disease.