Biology subjects

Mendoza, J. L.

Publications and source records attributed to Mendoza, J. L..

7 recordsLinked to original sources

bioRxiv2026

Graded and cross-reactive JAK-receptor interactions broaden the one-receptor, one-JAK rule

Cytokine receptors signal through a dedicated Janus kinase (JAK) that is thought to be constitutively bound to the receptor intracellular domain (ICD). We tested this one-receptor, one-JAK model biophysically. A bead-based screen of JAK1 and TYK2 against 32 receptor ICDs, confirmed by microscale thermophoresis to measure affinity, revealed dissociation constants spanning tens of nanomolar to tens of micromolar, indicating that many interactions are not high-affinity and may not be constitutive. Binding was also widely cross-reactive: GMCSFR bound both JAK1 and TYK2, and IL2R{beta} bound TYK2 far more tightly than JAK1. In cells, GM-CSF signaled through JAK1 when JAK2, JAK3, and TYK2 were inhibited. JAK-receptor pairing is therefore permissive and graded rather than exclusive and constitutive.

biochemistry↗

bioRxiv2026

High-Throughput Screening Identifies Proteasome Inhibitors as Potentiators of Type III Interferon JAK-STAT Signaling and Antiviral Immunity

Type III interferon (IFN3, IFN{lambda}) drives antiviral defense at epithelial barriers with a more favorable safety profile than Type I interferon (IFN1), but weaker signaling limits its therapeutic potential. To identify small molecules that potentiate IFN-JAK-STAT signaling, we developed a high-throughput live-cell assay measuring STAT1/STAT2 phosphorylation in A549 cells and screened ~8,640 compounds. This identified proteasome inhibitors (MG132, delanzomib, ixazomib, oprozomib, bortezomib) as selective potentiators of IFN3-induced STAT activation, with minimal effect on IFN1 signaling. Proteasome inhibition enhanced IFN3 antiviral activity to levels approaching IFN1. Mechanistically, proteasome inhibition increased cell-surface IL10R{beta}, the limiting IFN3 receptor subunit, thereby increasing receptor JAK-STAT signaling. It did not alter IFN1 receptor levels but protected IFNAR2 from late ligand-induced downregulation. These findings establish receptor subunit availability as a rate-limiting, druggable node governing IFN3 signaling strength and demonstrate a scalable platform for identifying cytokine-selective signaling potentiators, with implications for mucosal antiviral therapeutics.

immunology↗

bioRxiv2025

Geometric Tuning of Cytokine Receptor Association Modulates Synthetic Agonist Signaling

Cytokines signal by bringing receptor subunits together, but the role of receptor geometry in shaping signaling remains unclear because natural ligands enforce fixed assemblies. Here, we present a de novo protein design platform that rigidly scaffolds receptor-binding domains into defined spatial arrangements. Applying this across IL-7, type I and III interferons, IL-10, gp130, {beta} common, and synthetic receptor pairs, we show that by varying geometry, we can bias pSTAT pathway usage and tune functional outcomes. Geometric control allowed us to decouple pSTAT1 from pSTAT5 in IL-7, separate antigen presentation (MHC-I) from checkpoint induction (PD-L1) in type I interferons, and suppress pro-inflammatory IFN{gamma} secretion while retaining anti-inflammatory activity in IL-10. We further created minimal IL-6 and IL-3 agonists and strengthened synthetic receptor pairings inaccessible with present cytokines. These results establish receptor geometry as a central determinant of cytokine activity and provide a platform for programmable immune modulation.

synthetic biology↗

bioRxiv2022

Enhanced Complex Stability and Optimal JAK Geometry are Pivotal for a Potent Type III Interferon Response

Type I and III Interferons (IFN) constitute the host systems first line of defense against viral infections. Although the two families use distinct extracellular receptor complexes, an identical pair of Janus kinases (JAK) activates a similar set of signal transducer and activator of transcription (STATs) through a conserved pathway. Consequently, type I and III IFNs are presumed to activate a largely overlapping set of IFN-stimulated genes (ISGs) and elicit similar biological responses. Therapeutically, type III IFNs are attractive alternatives to type I IFNs due to their innate tissue specificity and lower systemic toxicity. However, a major limitation of type III IFNs is the significantly lower potency of their physiological activities compared to type I IFNs. To evaluate the role of receptor geometry in IFN signaling, we engineered cell lines that express wild-type and mutant IFN{lambda} receptors (IFN{lambda}R1) with rotated intracellular registers with respect to the associated JAK1. Evaluation of downstream signaling and biological activity of type III IFNs in cells with varied JAK-JAK geometries uncovered variant receptors that result in potentiation of type III IFN signaling and all downstream activities. With the combined use of a high-affinity ligand to stabilize the extracellular receptor complex, we found that the optimization of the intracellular JAK-JAK geometry enhances the type III IFN antiviral and anti-proliferative activities by 2- and 3-logs, respectively. In addition to providing a molecular basis for the observed differences in potency between type I and III IFNs, this work provides deeper insights into broader cytokine signaling mechanisms and novel blueprints for modulating cytokine functions to develop next generation antiviral and anticancer therapeutics.

cell biology↗

bioRxiv2022

Structural insights into the formation of repulsive Netrin guidance complexes

Netrins can dictate attractive and repulsive responses during axon growth and cell migration, where presence of the receptor UNC-5 on target cells results in Netrin-mediated repulsion. Molecular details of Netrin-UNC-5 interactions and how they signal remain elusive. Here, we show that nematode UNC-5 is a heparin-binding protein, and the UNC-5-heparin affinity can be modulated using directed evolution or via rational design using our novel structure of UNC-5 with a heparin fragment. Furthermore, UNC-5 and nematode UNC-6/Netrin form a large, stable and rigid oligomeric complex in the presence of heparin, which can incorporate the attractive UNC-40/DCC receptor, demonstrating binary and ternary ectodomain complexes at preparative scale. C. elegans with a heparin-binding deficient UNC-5 fail to establish proper gonad morphology due to abrogated distal tip cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Our findings establish Netrin responses to be mediated through glycosaminoglycan-regulated large macromolecular complexes.

biochemistry↗

bioRxiv2020

Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

genetics↗

bioRxiv2017

A Polymorphic Residue That Attenuates Interferon Lambda 4 Activity in Hominid Lineages

As antimicrobial signalling molecules, type III or lambda interferons (IFN{lambda}s) are critical for defence against infection by diverse pathogens. Counter-intuitively, expression of one member of the family, IFN{lambda}4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural in-frame nucleotide insertion that abrogates IFN{lambda}4 production improves viral clearance. To further understand how genetic variation between and within species affects IFN{lambda}4 function, we screened a panel of extant coding variants of human IFN{lambda}4 and identified three variants that substantially affect antiviral activity (P70S, L79F and K154E). The most notable variant was K154E, which enhanced in vitro activity in a range of antiviral and interferon stimulated gene (ISG) assays. This more active E154 variant of IFN{lambda}4 was found only in African Congo rainforest Pygmy hunter-gatherers. Remarkably, E154 was highly conserved as the ancestral residue in mammalian IFN{lambda}4s yet K154 is the dominant variant throughout evolution of the hominid genus Homo. Compared to chimpanzee IFN{lambda}4, the human orthologue had reduced activity due to amino acid substitution of glutamic acid with lysine at position 154. Meta-analysis of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFN{lambda}4 is consistent with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data suggest that human-specific K154 likely affects IFN{lambda}4 activity by reducing secretion and potency. We postulate that evolution of an IFN{lambda}4 with attenuated activity in humans (K154) likely contributes to distinct host-specific responses to and outcomes of infection, such as HCV.

immunology↗