Biology subjects

Blacklow, S. C.

Publications and source records attributed to Blacklow, S. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis for tetraspanin-dependent surface export and adhesive function of integrin α3β1

Integrin 3{beta}1 (ITG3{beta}1) is a member of an integrin subfamily that binds to laminin proteins and promotes attachment of epithelial cells to the basement membrane. ITG3{beta}1 forms a complex with the tetraspanin CD151, and loss-of-function mutations in both ITG3 and CD151 cause epidermolysis bullosa, a severe skin blistering disease resulting from a defect in basement membrane attachment. Here, we report the cryoEM structure of an ITG3{beta}1 complex with CD151 and show that mutation of CD151 at the binding interface disrupts complex formation in cells. Strikingly, CRISPR-mediated knockout of CD151 leads to a variably penetrant ITG3{beta}1 surface export defect that is restored by re-expression of wild-type but not interface-mutated CD151. Together, these studies define the molecular basis for binding of CD151 to ITG3{beta}1, and show that CD151 promotes ITG3{beta}1 surface export, providing a biochemical explanation for the CD151 loss-of-function phenotype in epidermolysis bullosa.

biochemistry↗

bioRxiv2026

High-throughput biochemical phenotyping of SHP2 variants reveals molecular basis of diseases and allosteric drug inhibition

Interpreting clinical and functional consequences of genetic variants remains challenging due to limited quantitative biochemical data at scale. We applied high-throughput microfluidic enzyme kinetics to profile 190 clinical variants of SHP2, a protein tyrosine phosphatase linked to developmental disorders and cancers. Through >300,000 reaction progress curves, we derived kinetic and thermodynamic parameters quantifying variant effects on catalysis, autoinhibition, stability, phosphopeptide binding, and drug responses. This multidimensional dataset reveals that dysregulated autoinhibition, rather than altered stability or catalysis, predominantly determines SHP2-associated pathogenesis. Thermodynamic modeling reveals that clinical-stage allosteric inhibitors preferentially stabilize a previously underappreciated, partially active conformation over the fully inactive state, leading to variant-dependent drug responses. Our high-throughput biochemical framework establishes a general approach to decipher the biochemical logic connecting protein variants to clinical outcomes.

biochemistry↗

bioRxiv2025

β-arrestin recruitment facilitates a direct association with G proteins

G protein-coupled receptors (GPCRs) are targets for almost a third of all FDA-approved drugs. GPCRs are known to signal through both heterotrimeric G proteins and {beta}-arrestins. Traditionally these pathways were viewed as largely separable, with G proteins primarily initiating downstream signaling while {beta}-arrestins modulate receptor trafficking and desensitization in addition to regulating their own signaling events. Recent studies suggest an integrated role of G proteins and {beta}-arrestins in GPCR signaling, however the cellular and biochemical requirements for G protein: {beta}-arrestin interactions remain unclear. Here we show that G proteins and {beta}-arrestins can directly interact. Through utilization of {beta}-arrestin-biased receptors and artificially enforced {beta}-arrestin relocalization, we demonstrate that recruitment of {beta}-arrestin to the plasma membrane is sufficient to interact with the G protein Gi. Using purified proteins, we show that Gi directly interacts with {beta}-arrestin. In addition, we find that Gi family members differ in their degree of association with {beta}-arrestin, and that a large degree of this selectivity resides within the alpha helical domain of Gi. These findings delineate the cellular and biochemical conditions that drive direct interactions between G proteins and {beta}-arrestins and illuminate the molecular basis for how they work together to effect GPCR signaling.

biochemistry↗

bioRxiv2024

The follicular lymphoma and chronic lymphocytic leukemia proliferative microenvironment at single-cell resolution

Adaptive immune responses occur lymph nodes (LNs) in a microenvironment established by resident stromal cells. LNs are also a site of proliferation of chronic lymphocytic leukemia (CLL), a B cell cancer that alters LN structure in a stereotypic manner. To deeply characterize reactive and CLL LNs, we developed a single-cell RNA sequencing pipeline. We find that proliferation of CLL cells in proliferation centers (PCs), a CLL-specific niche, begins with transient upregulation of MYC, subsequent downregulation of which may limit CLL growth. PCs contain a distinct fibroblast population expressing CCL19 while CLL cells express the CCL19 receptor CCR7, providing a recruitment mechanism for CLL cells to PCs. Using informatic, spatial, and in situ analyses to identify ligand-receptor pairs involving PC CLL cells and nearby immune and stromal cells, we observe that PCs are enriched for macrophages expressing BAFF, the integrin X{beta}2 heterodimer, and Galectin9, factors implicated in cell growth, adhesion, and immunosuppression. The most common predicted interactions in PCs involve CD74 and ligands such as MIF, and we find that CD74 blockade consistently inhibits CLL cell growth in culture. Our work highlights key features of the CLL proliferative niche and provides a roadmap for identifying vulnerabilities and new therapeutic strategies.

pathology↗

bioRxiv2024

Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examined the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction, through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

genomics↗

bioRxiv2024

Structural Requirements for Activity of Mind bomb1 in Notch Signaling

Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2[~]Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region of MIB1 (ccRING3) drives MIB1 dimerization and that ubiquitin transfer activity of MIB1 relies solely on RING3. We report x-ray crystal structures of a UbcH5B-ccRING3 complex as a fusion protein and of the ANK region. Directly tethering the N-terminal region to ccRING3 forms a minimal MIB1 protein, which is sufficient to induce a Notch response in receiver cells. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

biochemistry↗

bioRxiv2023

Molecular Mechanism of PP2A/B55α Inhibition by IER5

PP2A serine/threonine phosphatases are heterotrimeric complexes that execute many essential physiologic functions. These activities are modulated by additional regulatory proteins, such as ARPP19, FAM122A, and IER5. Here, we report the cryoelectron microscopy structure of a complex of PP2A/B55 with the N-terminal structured region of IER5 (IER5-N50), which occludes a surface on B55 used for substrate recruitment, and show that IER5-N50 inhibits PP2A/B55 catalyzed dephosphorylation of pTau in biochemical assays. Mutations of full-length IER5 that disrupt its PP2A/B55 interface interfere with co-immunoprecipitation of PP2A/B55. These mutations and deletions that remove the nuclear localization sequence of IER5 suppress cellular events such as KRT1 expression that depend on association of IER5 with PP2A/B55. Querying the Alphafold2 predicted structure database identified SERTA domain proteins as high-confidence PP2A/B55-binding structural homologs of IER5-N50. These studies define the molecular basis of PP2A/B55 inhibition by IER5-family proteins and suggest a roadmap for selective pharmacologic modulation of PP2A/B55 complexes.

biochemistry↗

bioRxiv2023

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. This process underlies nearly every aspect of proper B cell function. Here, we take advantage of peroxidase-catalyzed proximity labeling combined with quantitative mass spectrometry to track B cell co-receptor signaling dynamics from 10 seconds to 2 hours after BCR stimulation. This approach enables tracking of 2,814 proximity-labeled proteins and 1,394 quantified phosphosites and provides an unbiased and quantitative molecular map of proteins recruited to the vicinity of CD19, the key signaling subunit of the co-receptor complex. We detail the recruitment kinetics of essential signaling effectors to CD19 following activation, and then identify new mediators of B cell activation. In particular, we show that the glutamate transporter SLC1A1 is responsible for mediating rapid metabolic reprogramming immediately downstream of BCR stimulation and for maintaining redox homeostasis during B cell activation. This study provides a comprehensive map of the BCR signaling pathway and a rich resource for uncovering the complex signaling networks that regulate B cell activation.

immunology↗

bioRxiv2022

A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

molecular biology↗

bioRxiv2022

Structural Basis for Selective Proteolysis of ADAM10 Substrates at Membrane-Proximal Sites

The endopeptidase ADAM10 is a critical catalyst for regulated proteolysis of key drivers of mammalian development and physiology, and for non-amyloidogenic cleavage of the Alzheimers precursor protein as the primary -secretase. ADAM10 function in vivo requires formation of a complex with a C8-tetraspanin protein, with different ADAM10-C8-tetraspanin complexes having distinct substrate selectivity, yet the basis for such selectivity remains elusive. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and positions the enzyme active site about 20 [A] from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying selective ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

biochemistry↗

bioRxiv2022

Tethered agonist activated ADGRF1 structure reveals molecular preference for Gq signaling

Adhesion G-Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to modulate cell signalling. We report here that ADGRF1 is the first class B GPCR shown to signal through all major G-protein classes and identify the structural basis for its Gq preference by cryo-EM. Our structure shows that Gq over Gs preference in ADGRF1 derives from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helices VII and VIII at the site of G recruitment. Gs signalling is also more sensitive to mutation of TA or binding site residues than Gq. Our work advances the understanding of aGPCR TA activation in molecular detail, identifying structural features that potentially explain preferential signal modulation.

biochemistry↗

bioRxiv2022

A Fusion Protein Platform for Analyzing Tethered Agonism in the Adhesion Family of G Protein-Coupled Receptors

Adhesion G Protein-Coupled Receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates or unmasks a highly conserved tethered agonist (TA) that acts as an intramolecular ligand to stimulate G protein coupling. However, a systematic, comprehensive examination of signaling has not been performed for all aGPCR family members. Here, we report a platform for profiling TA-dependent activities of aGPCRs in several different assays and apply it to all 33 human family members. Activity profiling identified a heterogeneity of responses among these aGPCRs, with only [~]50% showing robust, TA-dependent signals. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA-dependence of the cellular responses. The signaling information in this dataset is a comprehensive resource relevant for the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.

biochemistry↗

bioRxiv2022

Functional Comparison Between Endogenous and Synthetic Notch Systems

The Notch pathway converts receptor-ligand interactions at the cell surface into a transcriptional response in the receiver cell. In recent years, synthetic Notch systems (SynNotch) that respond to different inputs and transduce different transcriptional responses have been engineered. One class of synNotch systems uses antibody-antigen interactions at the cell surface to induce the proteolytic cleavage cascade of the endogenous Notch autoregulatory core and the consequent release of a synNotch intracellular domain (ICD), converting surface antigen detection into a cellular response. However, whereas the activation of endogenous Notch requires ubiquitylation and subsequent endocytosis of the ligand ICD, these synNotch systems do not seem to have such a requirement because the synNotch ligands completely lack an ICD. This observation raises questions about existing models for the Notch activation mechanism. Here, we test how different structural and biochemical factors affect the dependence of endogenous and synthetic Notch activation on ligand ICD. We compare the behavior of antibody-antigen synNotch (aa-synNotch) to that of endogenous Notch, and to a synNotch system that uses induced FKBP-FRB dimerization with rapamycin (ff-synNotch), which still requires a ligand ICD. We found that differences in receptor-ligand affinity, in the identity of the transmembrane domain, or in the presence or absence of extracellular EGF repeats cannot explain the differences in ligand ICD requirement that distinguishes aa-synNotch from endogenous Notch or ff-synNotch. These findings suggest that the aa-SynNotch systems bypass the ligand ICD requirement because antigen-antibody pairs are able to promote other adhesive cell-cell interactions that provide the mechanical tension needed for ligand activation.

molecular biology↗

bioRxiv2022

An affinity-matured DLL4 ligand for broad-spectrum activation and inhibition of Notch signaling

The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 (DLL4) with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, engages human and murine Notch receptors with 500- to 1000-fold increased affinity compared to wild-type human DLL4. DeltaMAX also potently activates human Notch in plate-bound, bead-bound, and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch activation in response to either Delta-like (DLL) or Jagged (Jag) ligands, highlighting its utility as both an agonist and antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in primary activated human T cells. Taken together, our data defines DeltaMAX as a versatile biotechnological tool for broad-spectrum activation or inhibition of Notch signaling.

bioengineering↗

bioRxiv2021

Targeted degradation of the oncogenic phosphatase SHP2

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras/MAPK pathway upon stimulation of receptor tyrosine kinases (RTKs), which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at sub-micromolar concentration, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

biochemistry↗

bioRxiv2020

Design of Biologically Active Binary Protein 2D Materials

Proteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9-12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems. One Sentence SummaryCo-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

synthetic biology↗

bioRxiv2020

IER5, a DNA-damage response gene, is required for Notch-mediated induction of squamous cell differentiation

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, including IER5, which is regulated by Notch through several enhancer elements. We show that IER5 is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic to PPP2R2A, which encodes the B55 subunit of PP2A, and IER5 interacts with B55 in cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers. ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

cancer biology↗

bioRxiv2019

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking

CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 is a single-pass transmembrane protein belonging to the extensively studied Ig superfamily, CD81 belongs to a conserved but poorly understood family of four-pass transmembrane proteins called tetraspanins. These functionally diverse proteins play important roles in a wide variety of different organ systems by controlling protein trafficking and other cellular processes. Here, we show that CD81 relies on its ectodomain to control trafficking of CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is in complex with CD19. Mutations of CD81 in this contact interface suppress its CD19 surface-export activity. Taken together, these data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation, suggesting that this dynamism can be exploited to regulate B cell function. These results are not only important for understanding B cell biology, but also have important implications for understanding tetraspanin function more generally.

immunology↗