A Spatiotemporal Map of Co-Receptor Signaling Networks Underlying B Cell Activation

The B cell receptor (BCR) signals together with a multi-component co-receptor complex to initiate B cell activation in response to antigen binding. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track co-receptor signaling dynamics in Raji cells from 10 s to 2 h after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the signaling subunit of the co-receptor complex. We detail the recruitment kinetics of signaling effectors to CD19 and identify previously uncharacterized mediators of B cell activation. We show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of BCR signaling and a rich resource for uncovering the complex signaling networks that regulate activation

High-efficacy subcellular micropatterning of proteins using fibrinogen anchors.

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology

A grid-enabled web service for low-resolution crystal structure refinement

The deformable elastic network (DEN) method for reciprocal-space crystallographic refinement improves crystal structures, especially at resolutions lower than 3.5 Å. The DEN web service presented here intends to provide structural biologists with access to resources for running computationally intensive DEN refinements

Conformational Locking upon Cooperative Assembly of Notch Transcription Complexes

SummaryThe Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The “RAM” region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired-site DNA

The common oncogenomic program of NOTCH1 and NOTCH3 signaling in T-cell acute lymphoblastic leukemia

Notch is a major oncogenic driver in T cell acute lymphoblastic leukemia (T-ALL), in part because it binds to an enhancer that increases expression of MYC. Here, we exploit the capacity of activated NOTCH1 and NOTCH3 to induce T-ALL, despite substantial divergence in their intracellular regions, as a means to elucidate a broad, common Notch-dependent oncogenomic program through systematic comparison of the transcriptomes and Notch-bound genomic regulatory elements of NOTCH1- and NOTCH3-dependent T-ALL cells. ChIP-seq studies show a high concordance of functional NOTCH1 and NOTCH3 genomic binding sites that are enriched in binding motifs for RBPJ, the transcription factor that recruits activated Notch to DNA. The interchangeability of NOTCH1 and NOTCH3 was confirmed by rescue of NOTCH1-dependent T-ALL cells with activated NOTCH3 and vice versa. Despite remarkable overall similarity, there are nuanced differences in chromatin landscapes near critical common Notch target genes, most notably at a Notch-dependent enhancer that regulates MYC, which correlates with responsiveness to Notch pathway inhibitors. Overall, a common oncogenomic program driven by binding of either Notch is sufficient to maintain T-ALL cell growth, whereas cell-context specific differences appear to influence the response of T-ALL cells to Notch inhibition

A Spatiotemporal Notch Interaction Map From Plasma Membrane to Nucleus

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity

Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors

Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation

Biased Multicomponent Reactions to Develop Novel Bromodomain Inhibitors

BET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains

This is the author accepted manuscript. The final version is available from American Chemical Society via the DOI in this recordBromodomains have been pursued intensively over the past several years as emerging targets for the devel-opment of anti-cancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected poly-pharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selec-tive target profile is desired. Here we report that benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones, versatile ATP-site di-rected kinase pharmacophores utilized in the development of inhibitors of multiple kinases including a number of previ-ously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity as well as how to di-rect selectivity towards inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first report-ed kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers rec-ognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid dock-ing studies.This work was supported by NIH (Grant No. U54HL127365, to N.S.G. and J.W.; No. NIH P50 GM107618, to X.X. and S.C.B.; Nos. NIH U54 HD093540 and P01 CA066996, to J.Q.), the Medical Research Council (No. MC_UU_12016/2, to D.R.A.), the Spanish Ministerio de Economia y Competitividad (MINECO) (Grant No. SAF2015-60268R, to J.M.L.), and Fondo Europeo de Desarrollo Regional (FEDER) funds (to J.M.L.). D.L.B. was supported as a Merck Fellow of Damon Runyon Cancer Research Foundation (No. DRG-2196-14)

Notch and MAML-1 Complexation Do Not Detectably Alter the DNA Binding Specificity of the Transcription Factor CSL

Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined.Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes