Biology subjects

Garcia, K. C.

Publications and source records attributed to Garcia, K. C..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

PI3K regulates TAZ/YAP and mTORC1 axes that can be synergistically targeted

Abstract/SummaryO_ST_ABSPurposeC_ST_ABSSarcomas are a heterogeneous group of cancers with few shared therapeutic targets. PI3K signaling is activated in various subsets of sarcomas, representing a shared oncogenic signaling pathway. Oncogenic PI3K signaling has been challenging to target therapeutically. An integrated view of PI3K and Hippo pathway signaling is examined to determine if this could be leveraged therapeutically. Experimental designA tissue microarray containing sarcomas of various histological types was evaluated for PTEN loss and correlated with levels of activated TAZ and YAP. PI3K and Hippo pathways were dissected in sarcoma cell lines. The role of TAZ and YAP were evaluated in a PI3K-driven mouse model. The efficacy of mTORC1 inhibition and TEAD inhibition were evaluated in sarcoma cell lines and in vivo. ResultsPI3K signaling is frequently activated in sarcomas due to PTEN loss (in 30-60%), representing a common therapeutic target. TAZ and YAP are transcriptional co-activators regulated by PI3K and drive a transcriptome necessary for tumor growth in a PI3K-driven sarcoma mouse model. Combination therapy using IK-930 (TEAD inhibitor) and everolimus (mTORC1 inhibitor) synergistically diminished proliferation and anchorage independent growth of PI3K-activated sarcoma cell lines at low, physiologically achievable doses. Furthermore, this combination therapy showed a synergistic effect in vivo, reducing tumor proliferation and size. ConclusionsTAZ and YAP are transcriptional co-activators downstream of PI3K signaling, a pathway that has lacked a well-defined oncogenic transcription factor. This PI3K-TAZ/YAP axis exists in parallel to the known PI3K-Akt-mTORC1 axis allowing for synergistic combination therapy targeting the TAZ/YAP-TEAD interaction and mTORC1 in sarcomas.

cancer biology↗

bioRxiv2025

IL-9 as a naturally orthogonal cytokine with optimal JAK/STAT signaling for engineered T cell therapy

Arming T cells with a synthetically orthogonal IL-9 receptor (o9R) permits facile engraftment and potent anti-tumor functions. We considered whether the paucity of natural IL-9R expression could be exploited for T cell immunotherapy given that, in mice, high doses of IL-9 were well-tolerated without discernible immune modulation. Compared to o9R, T cells engineered with IL-9R exhibit superior tissue infiltration, stemness, and anti-tumor activity. These qualities are consistent with a stronger JAK/STAT signal, which in addition to STAT1/3/5, unexpectedly includes STAT4 (canonically associated with IL-12 but not common {gamma}-chain cytokines). IL-9R T cells are exquisitely sensitive to perturbations of proximal signaling, including structure-guided attenuation, amplification, and rebalancing of JAK/STAT signals. Biased IL-9R mutants uncover STAT1 as a rheostat between proliferative stem-like and terminally differentiated effector states. In summary, we identify native IL-9/IL-9R as a natural cytokine-receptor pair with near-orthogonal qualities and an optimal JAK/STAT signaling profile for engineered T cell therapy.

immunology↗

bioRxiv2024

De novo design and structure of a peptide-centric TCR mimic binding module

T cell receptor (TCR) mimics offer a promising platform for tumor-specific targeting of peptide-MHC in cancer immunotherapy. Here, we designed a de novo -helical TCR mimic (TCRm) specific for the NY-ESO-1 peptide presented by HLA-A*02, achieving high on-target specificity with nanomolar affinity (Kd = 9.5 nM). The structure of the TCRm/pMHC complex at 2.05 [A] resolution revealed a rigid TCR-like docking mode with an unusual degree of focus on the up-facing NY-ESO-1 side chains, suggesting the potential for reduced off-target reactivity. Indeed, a structure-informed in silico screen of 14,363 HLA-A*02 peptides correctly predicted two off-target peptides, yet our TCRm maintained a wide therapeutic window as a T cell engager. These results represent a path for precision targeting of tumor antigens with peptide-focused -helical TCR mimics.

immunology↗

bioRxiv2024

Potent antitumor activity of a designed interleukin-21 mimic

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNF blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon- (IFN-) and granzyme B compared to native IL-21, while increasing the frequency of IFN-+ Th1 cells and reducing that of Foxp3+ Tregs. As 21h10 has full human/mouse cross-reactivity, high stability and potency, and potentiates low-affinity antitumor responses, it has considerable translational potential.

biochemistry↗

bioRxiv2024

Design of high specificity binders for peptide-MHC-I complexes

Class I MHC molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance, and specific targeting of these peptide-MHC (pMHC) complexes could have considerable utility for treating diseases. Such targeting is challenging as it requires readout of the few outward facing peptide antigen residues and the avoidance of extensive contacts with the MHC carrier which is present on almost all cells. Here we describe the use of deep learning-based protein design tools to de novo design small proteins that arc above the peptide binding groove of pMHC complexes and make extensive contacts with the peptide. We identify specific binders for ten target pMHCs which when displayed on yeast bind the on-target pMHC tetramer but not closely related peptides. For five targets, incorporation of designs into chimeric antigen receptors leads to T-cell activation by the cognate pMHC complexes well above the background from complexes with peptides derived from proteome. Our approach can generate high specificity binders starting from either experimental or predicted structures of the target pMHC complexes, and should be widely useful for both protein and cell based pMHC targeting.

immunology↗

bioRxiv2024

Long-term single molecule localization microscopy uncovers dynamic co-assembly of Lrp6 and Ror2 into Wnt-signalosomes

The conserved Wnt signaling has been classified as two categories of canonical and noncanonical Wnt signaling. With a high promiscuity of Wnt signaling, how receptors from the two distinct pathways re-arrange in multi-protein signalosomes remains elusive. We here developed single-molecule tracking and localization microscopy based on labeling with reversibly binding nanobodies (rbTALM) for imaging receptor dynamics in the plasma membrane for extended time periods. To this end, we engineered nanobody-tag pairs with fine-tuned binding stabilities ensuring single-molecule tracking with high fidelity, yet continuous exchange of photobleached labels. Multicolor rbTALM imaging enabled simultaneous tracking and super-resolution imaging of three different Wnt co-receptors in the same cell for more than one hour at video rate. Time-lapse correlation analyses uncovered cooperative association of canonical and noncanonical Wnt co-receptors into a common, hybrid Wnt signalosome, demonstrating the exciting possibilities of rbTALM imaging for exploring nanoscale dynamics across millisecond to hour timescales.

biophysics↗

bioRxiv2024

A general platform for targeting MHC-II antigens via a single loop

Class-II major histocompatibility complexes (MHC-IIs) are central to the communications between CD4+ T cells and antigen presenting cells (APCs), but intrinsic structural features associated with MHC-II make it difficult to develop a general targeting system with high affinity and antigen specificity. Here, we introduce a protein platform, Targeted Recognition of Antigen-MHC Complex Reporter for MHC-II (TRACeR-II), to enable the rapid development of peptide-specific MHC-II binders. TRACeR-II has a small helical bundle scaffold and uses an unconventional mechanism to recognize antigens via a single loop. This unique antigen-recognition mechanism renders this platform highly versatile and amenable to direct structural modeling of the interactions with the antigen. We demonstrate that TRACeR-II binders can be rapidly evolved across multiple alleles, while computational protein design can produce specific binding sequences for a SARS-CoV-2 peptide of unknown complex structure. TRACeR-II sheds light on a simple and straightforward approach to address the MHC peptide targeting challenge, without relying on combinatorial selection on complementarity determining region (CDR) loops. It presents a promising basis for further exploration in immune response modulation as well as a broad range of theragnostic applications.

bioengineering↗

bioRxiv2023

IL-2-induced Stat3 Signaling is Critical for Effector Treg Cell Programming

Maintenance of immune homeostasis to the intestinal mictrobiota is dependent on a population of effector regulatory T (eTreg) cells that develop from microbiota-reactive induced (i)Treg cells. A cardinal feature of eTreg cells is their production of IL-10, which plays a non-redundant role in immune tolerance of commensal microbes. Here, we identify an unexpected role for IL-2-induced Stat3 signaling to program iTreg cells for eTreg cell differentiation and Il10 transcriptional competency. IL-2 proved to be both necessary and sufficient for eTreg cell development - contingent on Stat3 output of the IL-2 receptor coordinate with IL-2 signaling during early Treg cell commitment. Induction of iTreg cell programming in absence of IL-2-induced Stat3 signaling resulted in impaired eTreg cell differentiation and a failure to produce IL-10. An IL-2 mutein with reduced affinity for the IL-2R{gamma} ({gamma}c) chain was found to have blunted IL-2R Stat3 output, resulting in a deficiency of Il10 transcriptional programming that could not be fully rescued by Stat3 signaling subsequent to an initial window of iTreg cell differentiation. These findings expose a heretofore unappreciated role of IL-2 signaling that acts early to program subsequent production of IL-10 by developing eTreg cells, with broad implications for IL-2-based therapeutic interventions in immune-mediated diseases.

immunology↗

bioRxiv2023

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

biochemistry↗

bioRxiv2023

Rapid and accurate deorphanization of ligand-receptor pairs using AlphaFold

Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying novel ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold as a screening approach to identify extracellular ligand-binding pairs using a structural library of single-pass transmembrane receptors. Key to the approach is the optimization of AlphaFold input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. Importantly, the predictions were performed on ligand-receptor pairs not used for AlphaFold training. We demonstrate high discriminatory power and a success rate of close to 90 % for known ligand-receptor pairs and 50 % for a diverse set of experimentally validated interactions. These results demonstrate proof-of-concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

biochemistry↗

bioRxiv2023

FOXP3+ regulatory T cells use heparanase to access IL-2 bound to ECM in inflamed tissues

FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity. One-Sentence SummaryRegulatory T cells use heparanase to strip IL-2 bound to extracellular matrix within inflamed tissues, thereby supporting their homeostasis and function.

immunology↗

bioRxiv2023

Structural insight into guanylyl cyclase receptor hijacking of the kinase-Hsp90 regulatory mechanism

Membrane receptor guanylyl cyclases play a role in many important facets of human physiology, from regulating blood pressure to intestinal fluid secretion. The structural mechanisms which influence these important physiological processes have yet to be explored. We present the 3.9 [A] resolution cryo-EM structure of the human membrane receptor guanylyl cyclase GC-C in complex with Hsp90 and its co-chaperone Cdc37, providing insight into the mechanism of Cdc37 mediated binding of GC-C to the Hsp90 regulatory complex. As a membrane protein and non-kinase client of Hsp90-Cdc37, this work shows the remarkable plasticity of Cdc37 to interact with a broad array of clients with significant sequence variation. Further, this work shows how membrane receptor guanylyl cyclases hijack the regulatory mechanisms used for active kinases to facilitate their regulation. Given the known druggability of Hsp90, these insights can guide the further development of membrane receptor guanylyl cyclase-targeted therapeutics and lead to new avenues to treat hypertension, inflammatory bowel disease, and other membrane receptor guanylyl cyclase-related conditions.

biochemistry↗

bioRxiv2022

Receptor Elimination by E3 Ubiquitin Ligase Recruitment (REULR): A Targeted Protein Degradation Toolbox

In recent years, targeted protein degradation (TPD) of plasma membrane proteins by hijacking the ubiquitin proteasome system (UPS) or the lysosomal pathway have emerged as novel therapeutic avenues in drug development to address and inhibit canonically difficult targets. While TPD strategies have been successful to target cell surface receptors, these approaches are limited by the availability of suitable binders to generate heterobifunctional molecules. Here, we present the development of a nanobody (VHH) based degradation toolbox termed REULR (Receptor Elimination by E3 Ubiquitin Ligase Recruitment). We generated human and mouse cross-reactive nanobodies against 5 transmembrane PA-TM-RING type E3 Ubiquitin Ligases (RNF218, RNF130, RNF167, RNF43, ZNRF3) covering a broad range and selectivity of tissue expression with which we characterized expression in human and mouse cell lines and immune cells (PBMCs). We demonstrate that heterobifunctional REULR molecules can enforce transmembrane E3 ligase interaction with a variety of disease relevant target receptors (EGFR, EPOR, PD-1) by induced proximity resulting in effective membrane clearance of the target receptor at varying levels. In addition, we designed E3 Ligase self-degrading molecules, fratricide REULRs (RNF128, RNF130, RENF167, RNF43, ZNRF3), that allow downregulation of one or several E3 Ligases from the cell surface and consequently modulate receptor signaling strength. REULR molecules represent a VHH-based, modular and versatile "mix and match" targeting strategy for facile modulation of cell surface proteins by induced proximity to transmembrane PA-TM-RING E3 ligases.

bioengineering↗

bioRxiv2022

Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for co-receptor discrimination

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled receptors together with the shared LRP5/6 co-receptors to initiate {beta}-catenin signaling. The structure of a ternary complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of co-receptor discrimination by canonical Wnts by means of their N-termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker grafts were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

biophysics↗

bioRxiv2022

Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism

Natural Killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus infected cells, limiting their antitumor and antiviral activity. Here, we find that single-chain mouse NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activity to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between mouse NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45-NKG2A and CD45-Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells and possibly CD45 segregation within the NK cell-target cell synapse. These engineered ligands uncover a mechanism for enhancing the activity of mouse NK and T cells that merits evaluation in the context of human NKR antagonist cancer immunotherapies.

bioengineering↗

bioRxiv2022

Identification of orphan ligand-receptor relationships using a cell-based CRISPRa enrichment screening platform.

Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ("orphans") and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIRs). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

cell biology↗

bioRxiv2022

Live cell nanodot arrays uncover synergistic co-condensation of Wnt signalodroplets

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, we have developed biofunctional nanodot arrays (bNDAs) to spatially control dimerization and clustering of cell surface receptors at nanoscale. High-contrast bNDAs with spot diameters of [~]300 nm were obtained by capillary nanostamping of BSA bioconjugates, which were subsequently biofunctionalized by reaction with tandem anti- GFP clamp fusions. We achieved spatially controlled assembly of active Wnt signalosomes at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, we observed co-recruitment of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

biophysics↗

bioRxiv2022

Structure of the IL-1 27 quaternary receptor signaling complex

Interleukin 27 (IL-27) is a heterodimeric cytokine that functions to constrain T cell-mediated inflammation and plays an important role in immune homeostasis. Binding of IL-27 to cell surface receptors IL-27R and gp130 results in activation of receptor-associated Janus Kinases and nuclear translocation of STAT1 and STAT3 transcription factors. Despite the emerging therapeutic importance of this cytokine axis in cancer and autoimmunity, a molecular blueprint of the IL-27 receptor signaling complex, and its relation to other gp130/IL-12 family cytokines, is currently unclear. We used cryogenic-electron microscopy (cryo-EM) to determine the quaternary structure of IL-27 (p28/Ebi3) bound to receptor subunits, IL-27R and gp130. The resulting 3.47 [A] resolution structure revealed a three-site assembly mechanism nucleated by the central p28 subunit of the cytokine. The overall topology and molecular details of this binding are reminiscent of IL-6 but distinct from related heterodimeric cytokines IL-12 and IL-23. These results indicate distinct receptor assembly mechanisms used by heterodimeric cytokines with important consequences for targeted agonism and antagonism of IL-27 signaling.

immunology↗