Biology subjects

Smit, M. J.

Publications and source records attributed to Smit, M. J..

15 recordsLinked to original sources

bioRxiv2026

CCL25 targeting by de novo protein binders differentially suppress CCR9 and ACKR4 activation and transform CCL25 into a biased agonist

Chemokines and their receptors mediate cell migration and coordinate immune responses, while dysregulation can lead to inflammation. Therapeutic modulation of the chemokine signaling axis has proven difficult. Most drug discovery efforts target the receptors, whereas natural regulatory mechanisms focus on the chemokines. Despite this insight, development of effective chemokine-directed modulators has remained elusive. Recent advances in de novo protein design offer an unprecedented opportunity to produce high-affinity binders that efficiently block protein-protein interactions. We implemented a computational workflow leveraging the BindCraft platform to generate miniprotein binders against CCL25, the chemokine ligand for the receptors CCR9 and ACKR4 and implicated in inflammatory bowel diseases. The unbiased development results in several miniproteins designed to block the receptor N-terminus from wrapping the chemokine and prevent productive engagement. Thus, these proteins suppress CCL25-mediated effector coupling and halt MOLT-4 lymphoblast migration. Another class of miniprotein, represented by VUP25111, is predicted to bind CCL25 along the chemokine {beta}1 strand and retained receptor binding. This complex inhibited arrestin recruitment to CCR9, but not to ACKR4, indicating receptor specificity. Additionally, G protein signaling through CCR9 was unimpeded by VUP25111, suggesting that the miniprotein biased the native balanced agonist towards G proteins. These results demonstrate the effectiveness of differentially targeting CCL25 to suppress CCR9 signaling and new tools to resolve the structural basis of chemokine receptor activation and bias.

biochemistry↗

bioRxiv2026

Atypical GPCR Activation Resolved by Nanobody Engineering

Abstract/ Summary ParagraphG protein-coupled receptors (GPCRs) are the largest family of clinically targeted proteins, yet most therapeutics target a narrow subset of structurally well-behaved receptors. The atypical chemokine receptor ACKR3 defies canonical models, displaying broad ligand recognition, high basal activity, and resistance to inhibition. Using engineered nanobodies, cryo-EM, NMR, and structure-guided pharmacology, we uncover an unconventional activation mechanism in ACKR3 that challenges established paradigms of GPCR activation. We find that receptor activity is controlled by changes in extracellular pocket volume rather than conformational rearrangements in conserved microswitches, and an expanded aromatic cluster at the intracellular transducer binding pocket stabilizes the active state. These findings redefine how GPCRs can be modulated and open new strategies for targeting pharmacologically intractable receptors.

biochemistry↗

bioRxiv2026

GPCR kinase 3 phosphorylates atypical chemokine receptor 4 independent of G proteins

Atypical chemokine receptors (ACKRs) indirectly mediate cell migration through chemokine scavenging, which generally requires phosphorylation by G protein-coupled receptor (GPCR) kinases (GRKs) to efficiently control chemokine levels. Despite not coupling G proteins, ACKR4 is preferentially modified by GRK3, a kinase dependent on active G protein subunits for membrane translocation and phosphorylation activity. Here we resolve the underlying mechanisms allowing ACKR4 to circumvent the G protein requirement for GRK3 function. Using live cell BRET assays, we confirm that ACKR4 is preferentially phosphorylated by the GRK2/3 kinase family and that both GRK recruitment and receptor phosphorylation occur in the absence of activated G proteins. Instead, the kinases are recruited directly by a unique acidic rich motif in the proximal receptor C-terminus which coordinates productive phosphorylation reactions. Mutations in this region severely attenuated kinase recruitment and phosphorylation. Productive phosphorylation reaction plays a substantial role in the G protein-independent mechanism and a kinase-dead GRK3 (KD-GRK3) has severely reduced recruitment to ACKR4. This was not observed for KD-GRK3 translocation to GPCRs that recruit the kinase in a G protein-dependent manner. Together, these findings suggest that ACKR4 directly coordinates GRK3 recruitment and phosphorylation, highlighting a uniquely evolved atypical mechanism to utilize GRK2/3 while bypassing G protein activation and thereby supporting efficient chemokine scavenging by the atypical receptor.

biochemistry↗

bioRxiv2025

Identification of allo- or orthosteric VHH/single-domain antibodies that enhance or block pathogen binding to Siglec-1 on dendritic cells

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/695420v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@e85819org.highwire.dtl.DTLVardef@1eff6b4org.highwire.dtl.DTLVardef@12daa92org.highwire.dtl.DTLVardef@11959e5_HPS_FORMAT_FIGEXP M_FIG C_FIG BackgroundSiglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni. However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. ResultsHere we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1+ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis-binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans-interactions with ligands. ConclusionIn conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.

immunology↗

bioRxiv2025

Plasma membrane rather than endosomal Gq signaling drives transcriptional activity by the viral chemokine receptor US28 in glioblastoma

US28 is a human cytomegalovirus-encoded chemokine receptor homologue that has high agonist-independent activity, internalizes constitutively, and plays an oncomodulatory role in glioblastoma. As G protein signaling was originally believed to strictly occur at the plasma membrane, it has been assumed that US28s constitutive Gq/11 signaling is mediated by a minor population at the plasma membrane. However, accumulating evidence shows that some GPCRs activate G proteins from intracellular organelles, such as endosomes. Importantly, endosomal rather than plasma membrane G protein signaling has been associated with transcriptional activity. Here, we demonstrate that the endosomal US28 population robustly activates Gq/11, and thus, provides the major contribution of Gq/11 signaling. Surprisingly, US28 signaling at the plasma membrane rather than from endosomes primarily drives upregulation of gene expression involved in cell proliferation and inflammatory responses that are associated with glioblastoma and cancer. Our findings highlight the crucial role of receptor signaling location in cellular responses.

cell biology↗

bioRxiv2024

Disruption of basal CXCR4 oligomers impairs oncogenic properties in lymphoid neoplasms

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remain elusive. Using a newly established nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and pro-survival signaling via changes in the phosphoproteome, indicating the existence of basal CXCR4-oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B-cells to venetoclax is of clinical relevance. Taken together, we established a new, non-canonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that selective targeting thereof has clinical potential. Significance statementClass A GPCRs, including the chemokine receptor CXCR4, can form oligomers, but their functional relevance remains poorly understood. This study provides evidence for the role of basal CXCR4 oligomers in lymphoid neoplasms, where they drive pro-survival signaling, migration, and tumor growth. We use a novel nanobody-based BRET method to demonstrate that endogenous CXCR4 constitutively oligomerizes in lymphoid cancer cells, correlating with receptor expression levels. Pharmacological disruption of these oligomers reduces tumor- associated signaling, impairs spheroid growth, and sensitizes patient-derived malignant cells to the apoptosis-inducing drug Venetoclax. Since CXCR4 is frequently overexpressed and potentially clustered in various malignancies, this work offers broader implications for enhancing treatment efficacy, overcoming drug resistance, and potentially reducing side effects across multiple cancer types.

biochemistry↗

bioRxiv2024

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

biochemistry↗

bioRxiv2024

CCR9 signal termination is governed by an arrestin-independent phosphorylation mechanism

The chemokine receptor CCR9 coordinates immune cell migration from the thymus to the small intestine along gradients of CCL25. Receptor dysregulation is associated with a variety of inflammatory bowel diseases such as Crohns and ulcerative colitis, while aberrant CCR9 overexpression correlates with tumor metastasis. Despite being an attractive therapeutic target, attempts to clinically antagonize CCR9 have been unsuccessful. This highlights the need for a deeper understanding of its specific regulatory mechanisms and signaling pathways. CCR9 is a G protein-coupled receptor (GPCR) and activates Gi and Gq pathways. Unexpectedly, live-cell BRET assays reveal only limited G protein activation and signaling is rapidly terminated. Truncating the receptor C-terminus significantly enhanced G protein coupling, highlighting the regulatory role of this domain. Signal suppression was not due to canonical arrestin-coordinated desensitization. Rather, removal of GPCR kinase (GRK) phosphorylation led to sustained and robust G protein activation by CCR9. Using site-directed mutagenesis, we identified specific phosphorylation patterns that attenuate G protein coupling. Receptor internalization does not correlate with G protein activation capabilities. Instead, CCR9 phosphorylation appeared to directly destabilize the interaction of G protein heterotrimers with the receptor. This interference could lead to rapid loss of productive coupling and downstream signaling as phosphorylation would effectively render the receptor incapable of G protein coupling. An arrestin-independent, phosphorylation-driven deactivation mechanism could complement arrestin-dependent regulation of other GPCRs and have consequences for therapeutically targeting these receptors.

biochemistry↗

bioRxiv2024

Physical interaction with Ephrin B1 promotes CXCR4 intracellular localization and oncogenic potential

Chemokine receptor 4 (CXCR4) is a member of the chemokine receptor exclusively activated by the chemokine CXCL12. While CXCR4 regulates numerous physiological processes associated with cell migration and embryogenesis, its overexpression has been involved in various cancer types. Studies suggest that intracellular CXCR4 expression rather than CXCR4-operated signaling underlies its pro-tumorigenic functions. Given the role of GPCR interacting proteins in their trafficking and subcellular localization, we characterized the CXCR4 interactome using an affinity purification coupled to mass spectrometry (AP-MS) strategy. The most abundant protein identified in the CXCR4 interactome is Ephrin B1, a member of the Ephrin protein family that shares several functions with CXCR4, such as the regulation of cell migration and proliferation. Further studies showed that interaction between CXCR4 and Ephrin B1 is direct and enhanced by treating cell with CXCL12. They also indicated that Ephrin B1 prevents CXCR4 N-glycosylation, decreases CXCR4 cell surface expression and consistently inhibits CXCL12-induced CXCR4 coupling to Gi1-3 and the recruitment of {beta}-arrestins 1 and 2. Conversely, Ephrin B1 signals to Erk1,2 through CXCR4 activation and mediates the decrease in Death Receptor 5 expression elicited by intracellular CXCR4. Collectively, these findings identify Ephrin B1 as a key mediator of CXCR4 tumorigenic signaling.

biochemistry↗

bioRxiv2023

Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this {beta}-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a BRET-based {beta}-arrestin2 recruitment assay VUF15485 acts as an ACKR3 agonist with high potency (pEC50 = 7.6) and shows a similar extent of receptor activation compared to CXCL12 when using a newly developed, FRET-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485 was tested against a (atypical) chemokine receptor panel (agonist and antagonist mode) and proves to be selective for ACKR3. VUF15485 was subsequently labeled with tritium at one of its methoxy groups affording [3H]VUF15485. The small-molecule agonist radioligand binds saturably and with high affinity to human ACKR3 (Kd = 8.2 nM). [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (RT < 2 min) for its binding to ACKR3. Displacement of [3H]VUF15485 binding to membranes of HEK293T cells, transiently expressing ACKR3, with a number of CXCR3, CXCR4 or ACKR3 small-molecule ligands confirmed the ACKR3 profile of the [3H]VUF15485 binding site. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose VUF15485 to bind in the major and the minor binding pocket of ACKR3.

pharmacology and toxicology↗

bioRxiv2023

Luminescence-based screening for extracellular vesicle release modulators reveals a role for PI4KIIIβ in exosome biogenesis upon lysosome inhibition

Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad- spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIII{beta} as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions.

cell biology↗

bioRxiv2021

Computationally designed GPCR quaternary structures bias signaling pathway activation

Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can now be readily characterized, receptor associations into quaternary structures are very challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting membrane receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed stabilities, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only a few specific dimer structures also recruited {beta}-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a conformational switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs {beta}-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.

biophysics↗

bioRxiv2021

The developmental stage of the medulloblastoma cell-of-origin restricts Hedgehog pathway usage and drug sensitivity

Sonic Hedgehog (SHH) medulloblastoma originates from the cerebellar granule neuron progenitor (CGNP) lineage that depends on Hedgehog signaling for its perinatal expansion. While SHH tumors exhibit overall deregulation of this pathway, they also show patient age-specific aberrations. To investigate if the developmental stage of the CGNP can account for these age-specific lesions, we analyzed developing murine CGNP transcriptomes and observed highly dynamic gene expression as function of age. Cross-species comparison with human SHH medulloblastoma showed partial maintenance of these expression patterns, and highlighted low primary cilium expression as hallmark of infant medulloblastoma and early embryonic CGNPs. This coincided with reduced responsiveness to upstream Shh pathway component Smoothened, while sensitivity to downstream components Sufu and Gli was retained. Together, these findings can explain the preference for SUFU mutations in infant medulloblastoma and suggest that drugs targeting the downstream SHH pathway will be most appropriate for infant patients.

cancer biology↗

bioRxiv2020

Targeting the latent human cytomegalovirus reservoir with virus specific nanobodies

Latent reservoirs of viral pathogens are significant barriers to eradication of these viruses. During latency, herpesviruses maintain their genome, with little gene expression, making latent infections refractory to current treatments targeting viral replication. In the case of human cytomegalovirus (HCMV), sporadic reactivation events are well controlled by the immune system. However, in immunocompromised or immunosuppressed individuals, HCMV reactivation often results in morbidity in solid organ and stem cell transplant patients. Clearance of the latent reservoir could lower the incidence and severity of HCMV-associated disease. Here, we develop a virus specific nanobody (VUN100b) that partially inhibits signaling of the viral receptor US28. VUN100b treatment partially reverses latency without fully reactivating the virus. Moreover, VUN100b treatment drives recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals. This study shows the potential of VUN100b as a therapy to clear the HCMV latent reservoir of transplant patients.

microbiology↗

bioRxiv2020

Monitoring ligand-induced changes in receptor conformation with NanoBiT conjugated nanobodies

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

pharmacology and toxicology↗