Biology subjects

Kufareva, I.

Publications and source records attributed to Kufareva, I..

At least 19 recordsLinked to original sources

bioRxiv2026

Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease

cAMP-dependent Protein Kinase A (PKA) is a master regulator of cell signaling involved in energy metabolism, synaptic plasticity, and stress response. Dysregulated PKA signaling is implicated in diseases including neurodegeneration and cancer. PKA catalytic activity is regulated by two nonredundant regulatory subunits, Type I (RI/RI{beta}) and Type II (RII/RII{beta}), whose divergent functions are not fully understood. We generated double-knockout (KO) cell lines of RI/RI{beta} and RII/RII{beta} subunits and performed multiplexed MS-based proteomic and phosphoproteomic profiling under basal and glucose-perturbed conditions. We found that RI and RII loss drives distinct, and often opposite, remodeling of the cellular proteome and phosphoproteome. While both mutants blunted metabolic flexibility to glycolytic stressors and stimuli, RI and RII KO cells exhibited elevated and depressed glycolytic signaling, respectively. Interestingly, RI KO increased the abundance and kinase activity of the PKA catalytic subunit C isoform, leading to an increase in PKA substrate phosphorylation, whereas RII KO decreased the abundance, kinase activity, and substrate phosphorylation by the catalytic subunit C{beta} isoform. Notably, one of the most differentially affected PKA sites between RI and RII KOs maps to Tau, whose hyperphosphorylation is a hallmark of Alzheimers disease. Loss of RI increased Tau phosphorylation, which was not only caused by increased PKA catalytic activity, but also a higher binding affinity of Tau to RII subunits on the negatively-charged flexible linker region. Overall, the present study demonstrates that PKA RI and RII subunits play nonredundant roles in modulating PKA activity, metabolic flexibility, and phospho-regulation of key disease-associated substrates such as Tau.

biochemistry↗

bioRxiv2026

Elevated conformational dynamics makes ACKR3 activation-prone and G protein-incompetent

The atypical receptor ACKR3 works together with the canonical chemokine receptor CXCR4 to drive cell migration along gradients of their shared agonist CXCL12. CXCR4 promotes chemotaxis by activating canonical G protein pathways and recruiting {beta}-arrestins. ACKR3 indirectly regulates CXCR4-mediated chemotaxis by scavenging CXCL12. Unlike canonical chemokine receptors, ACKR3 does not couple to G proteins and instead is 100% biased towards {beta}-arrestins. CXCR4 activation by CXCL12 is exquisitely sensitive to subtle changes in both receptor and ligand. By contrast, ACKR3 is activation-prone: it recruits {beta}-arrestins in response to many ligands and is much less sensitive to mutations, suggesting distinct activation mechanisms compared to CXCR4. To explore the basis of these differences, we compared the dynamics of ACKR3 and CXCR4 complexes with chemokines using molecular dynamic (MD) simulations. Ten-microsecond atomistic MD simulations revealed that CXCR4 adopts a stable active state when bound to WT CXCL12 but transitions to an inactive state when in complex with the antagonist variant, [P2G]CXCL12. By comparison, ACKR3 exhibits a variable transmembrane (TM) 6 state distribution and persistently "active" TM7 when complexed with either WT CXCL12 or [P2G]CXCL12, the latter retaining substantial agonistic activity at ACKR3. We further identified ligand-mediated residue interaction networks in the TM core that regulate TM6 and TM7 activation in CXCR4 but are absent or disrupted in ACKR3, resulting in less constrained receptor dynamics. These findings were validated by BRET-based assays with CXCL12 and ACKR3 mutants. Together, the data suggests that the unique conformational dynamics of ACKR3 govern its activation propensity, its ligand promiscuity, and its atypical effector coupling.

pharmacology and toxicology↗

bioRxiv2026

Pharmacological proximities in the GPCR family discovered using contact-informed amino-acid and binding pocket similarities

Understanding protein proximities in the theoretical ligand space is essential for developing therapeutics with desirable polypharmacology, predicting off-targets, and discovering surrogate ligands for poorly characterized proteins. This is especially important for G protein-coupled receptors (GPCRs) - a major class of drug targets, many of which still lack known ligands. Circumventing this limitation, we present GPCR-CoINPocket v2, a contact-informed metric for detecting GPCR pharmacological similarities from amino-acid sequences alone. We first establish a "gold standard" of pharmacological relatedness using ChEMBL-derived ligand sets. We then replace traditional evolutionary amino acid similarity matrices with a chemically-informed matrix derived from protein:ligand interaction patterns across 3,306 structures, significantly improving early detection of shared pharmacology between distantly homologous receptors. An additional unconstrained, contact-informed matrix further enhances predictive performance. Pilot application of the method revealed previously unrecognized similarities between the {beta}2 adrenoceptor and three Class A peptide GPCRs, which we confirmed experimentally by demonstrating the binding of select ligands of these receptors to the {beta}2. Dimensionality reduction of similarity scores recapitulates known receptor relationships and predicts neighbors of orphan GPCRs later confirmed experimentally. Overall, GPCR-CoINPocket v2 provides a powerful sequence-based framework to prioritize ligand space, predict polypharmacology, and accelerate GPCR drug discovery and deorphanization.

bioinformatics↗

bioRxiv2026

Monocyte Migration Emerges from a Divergent Chemokine Signaling Network

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

systems biology↗

bioRxiv2026

A NOD2-Encoded Toggle Switch Resolves the Host-Microbe Battle Over Cyclic AMP Control

Pathogens hijack macrophages by triggering pathological cAMP surges that block phagolysosomal killing--a defect mirrored in phagocytes from refractory colitis. We identify a host-encoded, pathogen-specific surge-protector comprised of a three-protein toggle: The innate sensor NOD2 binds and masks an evolutionarily conserved motif in GIV that activates trimeric-GTPase Gi, enforcing a biphasic surge-to-plunge cAMP-program: early, NOD2*GIV assembly permits a brief, tolerogenic cAMP rise, whereas subsequent GIV*Gi engagement collapses cAMP to drive phagolysosomal fusion and microbial clearance. Structural, biochemical, and ultrastructural analyses reveal how molecular toggling imposes precise spatial and temporal control. Pharmacogenomic perturbations pinpoint cAMP-PKA hyperactivation as the defining lesion in GIV-deficient macrophages. Functional studies in primary macrophages and human gut organoid co-cultures show that toggling the NOD2*GIV*Gi-axis is necessary and sufficient to convert tolerant macrophages into microbicidal machines that preserve mucosal barrier integrity. These findings uncover a druggable cAMP-control pathway with therapeutic promise in colitis. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/715116v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@53cd40org.highwire.dtl.DTLVardef@1c83c82org.highwire.dtl.DTLVardef@fcbc58org.highwire.dtl.DTLVardef@b14f8e_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC BlurbPathogens hijack macrophages by inducing cAMP surges that help them evade clearance. Anandachar et al. identify a host "toggle switch" in which NOD2 and G proteins compete for GIV, driving a rapid and robust surge-to-plunge transition in cAMP. This temporal switch limits tolerogenic signaling, restores microbial clearance and barrier integrity, and unveils a targetable host pathway in infection and IBD. HighlightsO_LIPathogens exploit cAMP surges in macrophages to block phagolysosomal killing of microbes C_LIO_LIGIV acts as a molecular "toggle" linking NOD2 sensing to Gi-mediated cAMP control C_LIO_LIStructural and mutagenesis studies reveal mutually exclusive binding of NOD2 and Gi to GIV C_LIO_LIPharmacogenomic perturbations pinpoint PKA, not EPAC, as the critical downstream effector C_LIO_LIOrganoid co-cultures show NOD2-GIV-PKA crosstalk safeguards microbial clearance and gut barrier integrity C_LI

immunology↗

bioRxiv2025

Signaling bias of the protease-activated receptor-1 is dictated by distinct GRK5 and β-arrestin-2 determinants

G protein-coupled receptors (GPCRs) exhibit signaling bias, or preferential activation of heterotrimeric G proteins versus GPCR receptor kinase (GRK)-mediated {beta}-arrestin signaling. The protease-activated receptor-1 (PAR1) GPCR activates both G protein and {beta}-arrestin in response to thrombin, but only {beta}-arrestin in response to activated protein C (APC). Thrombin-activated PAR1-G protein signaling is desensitized by {beta}-arrestin-1, whereas APC-activated PAR1 signaling is propagated by {beta}-arrestin-2. The mechanisms underlying PAR1 biased signaling are not known. Here, using computational modeling and cell biology studies, we reveal the molecular basis of signaling by thrombin- and APC-activated PAR1. Although both thrombin- and APC-induced PAR1 signaling are regulated by the same GRK isoform, GRK5, the two types of signaling are differentially dependent on GRK5 membrane anchoring, PAR1 C-terminal phosphorylation, and the binding mode of {beta}-arrestin-2. These differences translate into distinct {beta}-arrestin-2 conformations and define the cytoprotective signaling signature by APC which contrasts with thrombin inflammatory signaling.

cell biology↗

bioRxiv2025

Rules of engagement: determinants of chemokine receptor activation and selectivity by CCL27 and CCL28

The distinct functional roles of chemokines CCL27 and CCL28 in epithelial immunity of skin and mucosal tissues, respectively, are coordinated by their shared receptor, CCR10 and the CCL28-specific receptor, CCR3. In this study, we conducted structure-function studies focused on the N-termini of these two chemokines to identify determinants of receptor activation, internalization and binding specificity. Deletion of two N-terminal residues of CCL27 resulted in a CCR10 antagonist, highlighting the critical roles of these residues in driving receptor pharmacology. Extension with a Phe produced a superagonist by occupying an available subpocket in the receptor binding site. Swapping the N-terminus of CCL28 onto the CCL27 globular domain (NT28-CCL27) also resulted in a superagonist of CCR10, but the opposite swap (NT27-CCL28) showed equivalent or reduced activity compared to WT CCL28, indicating that the CCL28 N-terminus is a stronger driver of CCR10 signaling. The effect of these and other mutations were rationalized by AlphaFold models of the CCR10 complexes. AlphaFold modeling also revealed that the reduced size of the binding pocket, and more basic nature of the N-terminus and extracellular loops of CCR3 compared to CCR10, contribute to its specificity for CCL28 while CCR10 accommodates both ligands. The overall basic nature of CCL28 also contributes to its high affinity for glycosaminoglycans, which is likely important for its retention in mucosal tissues. These data illustrate the modular compositions of these chemokines that has evolved to achieve overlapping but non-redundant functions, and the exploitation of this modular nature to produce engineered chemokines for probing or targeting CCR10 in disease contexts.

pharmacology and toxicology↗

bioRxiv2025

The 4th GPCR Dock: assessment of blind predictions for GPCR-ligand complexes in the era of AlphaFold

The GPCR Dock competitions are a series of community-wide assessments of computational structural modeling and ligand docking for G protein-coupled receptors, a central class of drug targets in the human proteome. The assessments are designed to provide an unbiased overview of progress and to pinpoint areas in need of refinement, thus shaping and directing the development of computational modeling methodologies for GPCRs. In the footsteps of 2008, 2010, and 2013 assessments, the 4th round (GPCR Dock 2021) featured five diverse and challenging prediction targets and coincided with the emergence of AlphaFold, the revolutionary Artificial Intelligence (AI) technology for protein structure prediction from amino acid sequences. This report summarizes the assessment results and challenges in the context of the convergent evolution of computational and experimental structure determination techniques for GPCRs. We demonstrate that thanks to the breakthroughs in AI-powered modeling, the accuracy of modern computational models of GPCR complexes with peptides can not only approach but also exceed that of low-resolution experimental structures. However, our results highlight the unwavering need for high-resolution GPCR structure determination, especially with small molecule chemicals, and for the concurrent application of physics-based and expert-guided modeling methods.

molecular biology↗

bioRxiv2024

Essential strategies for the detection of constitutive and ligand-dependent Gi-directed activity of 7TM receptors using bioluminescence resonance energy transfer

The constitutive (ligand-independent) signaling of G protein-coupled receptors (GPCRs) is being increasingly appreciated as an integral aspect of their function; however, it can be technically hard to detect for poorly characterized, e.g. orphan, receptors of the cAMP-inhibitory Gi-coupled (GiPCR) family. In this study, we delineate the optimal strategies for the detection of such activity across several GiPCRs in two cell lines. As our study examples, we chose two canonical GiPCRs - the constitutively active Smoothened and the ligand-activated CXCR4,-and one atypical GPCRs, the chemokine receptor ACKR3. We verified the applicability of three Bioluminescence Resonance Energy Transfer (BRET)-based assays - one measuring changes in intracellular cAMP, another in G{beta}{gamma}/GRK3ct association and third in Gi-G{beta}{gamma} dissociation, - for assessing both constitutive and ligand-modulated activity of these receptors. We also revealed the possible caveats and sources of false positives, and proposed optimization strategies. All three types of assays confirmed the ligand-dependent activity of CXCR4, the controversial G protein incompetence of ACKR3, the constitutive Gi-directed activity of SMO, and its modulation by PTCH1. We also demonstrated that PTCH1 promotes SMO localization to the cell surface, thus enhancing its responsiveness not only to agonists but also to antagonists, which is a novel mechanism of regulation of a Class F GiPCR Smoothened.

pharmacology and toxicology↗

bioRxiv2024

Noncanonical roles of chemokine regions in CCR9 activation revealed by structural modeling and mutational mapping

The G protein-coupled CC chemokine receptor 9 (CCR9) plays a major role in inflammatory bowel disease and has been implicated in cancer. Despite its importance as a drug target, there is limited mechanistic understanding of how CCR9 engages and is activated by its endogenous chemokine agonist CCL25. Here, by combining structural modeling with multimodal pharmacological assessment of receptor mutants, we generated a functional map of the CCR9-CCL25 interaction interface and delineated key determinants of binding, agonism, constitutive activity, and G protein vs arrestin signaling. In contrast to all complexes studied to date, where chemokines drive receptor activation through their N-termini, we determined that CCL25 activates CCR9 via a distinct region, its 30s loop. In support of this non-canonical mechanism, CCR9 signaling is tolerant to alanine mutations in the N-terminus of CCL25 but strongly affected by modifications to the 30s loop. Through molecular evolution of the CCL25 N-terminus, we identified chemokine analogs with enhanced binding properties. However, in contrast to other receptor-chemokine systems, these analogs remained full agonists, consistent with the localization of CCL25 signaling determinants outside of the N-terminus. The non-canonical signature of CCR9 activation provides new insights to aid CCR9 drug discovery and may also inform structure-based design of drugs targeting other chemokine receptors.

molecular biology↗

bioRxiv2023

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

pharmacology and toxicology↗

bioRxiv2023

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics

CC chemokine receptor 5 (CCR5) promotes inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A CCR5 inhibitor has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of other diseases has had limited success, likely because of the complexity of CCR5 pharmacology and biology. CCR5 is activated by natural and engineered chemokines that elicit distinct signaling and trafficking responses, including receptor sequestration inside the cell. Intracellular sequestration may be therapeutically exploitable as a strategy for receptor inhibition, but the mechanisms by which different ligands promote receptor retention in the cell versus presence on the cell membrane are poorly understood. We employed live cell ascorbic acid peroxidase (APEX2) proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveal distinct ligand-dependent CCR5 trafficking patterns with temporal and spatial resolution. All three chemokines internalize CCR5 via {beta}-arrestin-dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the cellular mechanisms behind CCR5 intracellular sequestration and suggest how trafficking can be exploited for the development of functional antagonists of CCR5. Significance StatementCCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and transmission of HIV. It responds to different ligands with distinct signaling and trafficking behaviors; notably some ligands induce retention of the receptor inside the cell. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the cellular basis for receptor sequestration that can be exploited as a therapeutic strategy for inhibiting CCR5 function.

cell biology↗

bioRxiv2022

Phosphorylation of Gαi shapes canonical Gα(i)βγ/GPCR signaling

A long-standing question in the field of signal transduction is to understand the interplay between distinct signaling pathways that control cell behavior. For growth factors and heterotrimeric G proteins, the two major signaling hubs in eukaryotes, the mechanisms of independent signal transduction have been extensively characterized; however, if/how they may cross talk remains obscure. Here we use linear-ion-trap mass spectrometry in combination with cell-based biophysical, biochemical, and phenotypic assays to chart at least three distinct ways in which growth factors may impact canonical G(i){beta}{gamma} signaling downstream of a GPCR (CXCR4) via phosphorylation of Gi. Phosphomimicking mutations in a cluster of residues in the E helix (Y154/Y155) result in the suppression of agonist-induced G(i) activation while promoting constitutive G{beta}{gamma} signaling; others in the P-loop (Ser44, Ser47, Thr48) suppress Gi activation entirely thus completely segregating the growth factor and GPCR pathways. While most phosphoevents appear to impact, as expected, the core properties of G(i) (conformational stability, nucleotide binding, G{beta}{gamma} association and release, etc.), one phosphomimicking mutation promoted mislocalization of Gi from the plasma membrane: a novel and unexpected mechanism of GPCR signal suppression. A phosphomutation of C-terminal Y320 was sufficient to orchestrate such suppression by protein compartmentalization. Findings not only elucidate how growth factor and chemokine signals crosstalk through phosphomodulation of Gi, but also how such crosstalk may generate signal diversity.

cell biology↗

bioRxiv2020

Evolution of Modularity, Interactome and Functions of GIV/Girdin (CCDC88A) from Invertebrates to Vertebrates

PDZ domains are one of the most abundant protein domains in eukaryotes and frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and finetune cellular signaling. Here we describe the presence of a PBM on GIV/Girdin (CCDC88A) that is conserved throughout evolution, from invertebrates to vertebrates, and is generated as a long isoform-variant in humans, which we named GIV-L. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes to the cell junctions, and has a unique PDZ-interactome, which impacts GIV-Ls ability to bind and activate trimeric G-protein, Gi through its guanine-nucleotide exchange modulator (GEM) module; the GEM module is found exclusively in vertebrates. Thus, the two functional modules in GIV evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analyses in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how GIV/CCDC88A in humans displays evolutionary flexibility in modularity, which allows the resultant isoforms to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV).

biochemistry↗

bioRxiv2020

Orphan G protein-coupled receptor, GPR37L1: pharmacological toolbox empty once again

Orphan G protein-coupled receptors (GPCRs) are largely intractable therapeutic targets, owing to the lack of chemical tools for exploring their pharmacology. The discovery of such tools, however, is hampered by a number of unknowns, such as effector coupling and appropriate positive controls. In our 2017 Nature Chemical Biology paper1, we developed a computational chemical tool discovery approach called GPCR Contact-Informed Neighboring Pocket (GPCR-CoINPocket). This method predicted pharmacological similarity of GPCRs in a ligand- and structure-independent manner, to enable the discovery of off-target activities of known compounds at orphan GPCRs and hence the identification of so-called surrogate ligands. Our orphan GPCR target for prospective surrogate ligand discovery efforts was GPR37L1, a brain-specific receptor linked to cerebellar development2 and seizures3. We had previously demonstrated that GPR37L1 constitutively coupled to Gs and generated ligand-independent increases in intracellular cAMP4[§]. Thus, the inverse agonist activities of computationally predicted surrogates were tested in the cAMP response element luciferase (CRE-luc) reporter gene assay in human embryonic kidney (HEK293) cells expressing either vector control or what we thought was untagged GPR37L1 in pcDNA3.1. However, we recently discovered that the GPR37L1 construct used in that study was incorrect: instead of pcDNA3.1, it carried the receptor inserted backwards into a yeast p426GPD vector (hereafter referred to as p426-r37L1). Here, we correct the cloning error and describe our subsequent unsuccessful efforts to re-test the computationally predicted GPR37L1 ligands (triggering an author-initiated retraction of1). NoteWe, the authors, are working with the Nature Chemical Biology Editors to retract our 2017 paper Orphan receptor ligand discovery by pickpocketing pharmacological neighbors1. The present manuscript is under review at Nature Chemical Biology as a Matters Arising accompaniment to the anticipated author-initiated retraction. We initiated the steps towards the retraction upon discovering a regrettable cloning error that put into question the in vitro findings reported in1. This action was unanimously agreed upon by all authors. The computational aspects of the original manuscript1 are unaffected by this error.

pharmacology and toxicology↗

bioRxiv2020

TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multi-modular scaffold, GIV (a.k.a Girdin) titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (LPS, a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress pro-inflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene depletion studies confirmed that the presence of GIV ameliorates DSS-induced colitis and sepsis-induced death. The anti-inflammatory actions of GIV are mediated via its C-terminally located TIR-like BB-loop (TILL)-motif which binds the cytoplasmic TIR-modules of TLR4 in a manner that precludes receptor dimerization; the latter is a pre-requisite for pro-inflammatory signaling. Binding of GIVs TILL motif to other TIR modules inhibits pro-inflammatory signaling via other TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses. SignificanceTo ensure immunity, and yet limit pathology, inflammatory responses must be confined within the proverbial Goldilocks zone. TLR4 is the prototypical sensor that orchestrates inflammatory responses through a series of well characterized downstream cascades. How TLR4 signals are confined remain incompletely understood. Using trans-scale approaches ranging from disease modeling in live animals, through cell-based interventional studies, to structure-guided biochemical studies that offer an atomic-level resolution, this study unravels the existence of a brake within the TLR4 signaling cascade, i.e., GIV; the latter is a prototypical member of an emerging class of scaffold proteins. By showing that GIV uses conserved mechanisms to impact multi-TLR signaling, this work unravels a multi-scale point of convergence of immune signaling of broader impact beyond TLR4.

immunology↗

bioRxiv2020

Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs crosstalk has been a long-standing question, but answers remain elusive. Using linear-ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator, GIV, dissociates Gi*{beta}{gamma} trimers, scaffolds monomeric Gi with RTKs, and facilitates the phosphorylation on two tyrosines located within the inter-domain cleft of Gi. Phosphorylation triggers the activation of Gi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cells decision to go vs. grow. These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes. Significance StatementGrowth factors and heterotrimeric G proteins are two of the most widely studied signaling pathways in eukaryotes; their crosstalk shapes some of the most fundamental cellular responses in both health and disease. Although mechanisms by which G protein pathways transactivate growth factor RTKs has been well-defined, how the reverse may happen is less understood. This study defines the key steps and cellular consequences of a fundamental mechanism of signal crosstalk that enables RTKs to transactivate heterotrimeric G protein, Gi. Mutations found in tumors shed light on how derailing this mechanism impacts tumor cell behavior. Thus, findings not only show how cells integrate extracellular signals via pathway crosstalk, but also demonstrate the relevance of this pathway in cancers.

biochemistry↗

bioRxiv2020

Functional anatomy of the full length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis

Due to their prominent role in development and infamy in cancer and HIV, the chemokine receptor CXCR4 and its ligand, CXCL12, have been the subject of numerous structural and functional studies. Nevertheless, a high resolution structure of the CXCR4-CXCL12 complex has not been reported. Even with several alternative computational models of the complex at hand, the relative contributions of different interaction epitopes to ligand binding, ligand selectivity and signaling are not readily apparent. Here, building upon our latest structural model, we employed a systematic mutagenesis strategy to dissect the functional anatomy of the of CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments supported pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor, while also providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N-terminus to chemokine signaling; this finding challenges a longstanding "two-site" hypothesis about the essential features of the receptor-chemokine interaction where the N-terminus is purported to only contribute to binding affinity. The results suggest that while the interaction of the chemokine N-terminus with the receptor binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N-terminus binds the chemokine. Along with systematic characterization of other epitopes, the current data allow us to propose a comprehensive experimentally-consistent structural model for how the chemokine binds CXCR4 and initiates signal transmission through the receptor TM domain. One sentence summaryA systematic structure-guided mutagenesis study of chemokine receptor CXCR4 reveals novel insights into epitopes regulating ligand recognition, ligand specificity and CXCL12-mediated signaling.

pharmacology and toxicology↗