Biology subjects

Paulo, J. A.

Publications and source records attributed to Paulo, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Structures of MyD88 and its oncogenic mutant reveal DNA binding with implications for cytosolic DNA sensing

MyD88 is a universal adaptor that connects to Toll-like receptors and interleukin-1 receptors through its Toll/IL-1R homology (TIR) domain and activates inflammatory signaling via its death domain (DD). However, how MyD88TIR serves as the platform for these functions and if it has additional functions remain unclear. Here, we report cryogenic electron microscopy (cryo-EM) structures of MyD88TIR and its cancer-specific mutant L252P (previously L265P) mainly in ring and spiral forms, respectively. Both assemblies are formed by antiparallel double-filaments, using canonical TIR interfaces within each strand but unique surfaces between the strands, and position MyD88DD at the same sides of the assemblies for self-oligomerization and pathway activation. Surprisingly, these WT or L252P filaments are bound to dsDNA using the concave sides of the rings and the spirals. In vitro assays and cellular studies establish that MyD88 directly binds dsDNA and promotes inflammatory cytokine expression, particularly pro-IL-1{beta}, following dsDNA transfection or viral infection. Notably, these responses are unaffected by deficiency of UNC93B1, the essential chaperone required for trafficking endosomal nucleic acid-sensing TLRs. Structure-guide MyD88 mutant that disrupts dsDNA binding specifically compromises dsDNA-induced transcriptional program while maintaining TLR signaling. RNA-seq further reveals that MyD88 plays a crucial role in inducing gene expression upon dsDNA sensing and that about half of cGAS-dependent genes are also MyD88-dependent. Together, our structural, biochemical and genetic data support the findings that MyD88 directly engages with cytosolic dsDNA and cooperates with cGAS to promote the full innate immune transcriptional response.

immunology↗

bioRxiv2026

KRASG12C Inhibition Remodels the Non-Small Cell Lung Cancer Surfaceome to Uncover Targetable Vulnerabilities

Mutant-selective and pan-RAS(ON) multi-selective inhibitors have improved clinical outcomes for patients with RAS-driven cancers; however, adaptive resistance limits the depth and durability of response. Using an integrated genetic, transcriptomic, and mass-spectrometry-based quantitative temporal proteomic platform, we profile the adaptive response to KRASG12C inhibitors (KRASi) in KRASG12C-mutant lung adenocarcinoma models. Our analyses reveal remodeling of signaling pathways and the cell surface proteome, including cell surface proteins selectively upregulated across cell lines, tumor models, and treatment durations. Folate receptor alpha (FOLR1) emerged as a therapeutically actionable target that is significantly upregulated with KRASi and is part of a broader acute cellular state transition characterized by acquisition of an epithelial identity. Combining KRASi with mirvetuximab soravtansine-gynx, a clinically approved FOLR1-targeting antibody drug conjugate, enhanced tumor cell cytotoxicity and deepened response and tumor control in cellular and in vivo models. Additionally, this work represents a comprehensive multi-omic resource characterizing non-genetic temporal adaptation to KRASi for future mechanistic and therapeutic investigations.

cancer biology↗

bioRxiv2026

KRAS inhibition drives adaptive surfaceome remodeling and reveals ALPP/ALPPL2 as a targetable vulnerability in pancreatic cancer

KRAS is mutated in over 90% of pancreatic ductal adenocarcinoma (PDAC) and is required for tumor initiation and maintenance. While clinical trials testing mutant-selective and pan-RAS inhibitors (KRASi) have demonstrated significant clinical benefit, genetic and non-genetic mechanisms of resistance limit the long-term efficacy of monotherapy strategies. Here, we used quantitative temporal analysis of the global, phosphorylation, and surface proteome to comprehensively identify non-genetic adaptive pathways and cell surface biomarkers in response to acute and long-term KRAS inhibition in PDAC. Through integrative machine learning analysis of the KRASi-proteome and publicly available datasets, we discovered top priority cell surface targets for combination therapy with KRASi. We identified Placental Alkaline Phosphatase (ALPP) as upregulated in response to KRASi through compensatory JAK2/STAT3 signaling. We developed a novel clinical grade ALPP/ALPPL2 (ALPG)-targeting antibody drug conjugate (ADC) with site-specific MMAE conjugation and demonstrated single agent efficacy in PDAC models, as well as enhanced internalization and anti-tumor efficacy in combination with KRASi. Together, these studies comprehensively define the KRASi-induced proteome and identify ALPP/ALPPL2 as a promising cell surface target for combination with KRASi to enhance response and forestall KRASi resistance.

cancer biology↗

bioRxiv2026

Tumor suppressor-like mutations mark a dosage-constrained dependency in leukemia

Cancer genes are classically categorized as oncogenes or tumor suppressors based on whether gain or loss of function promotes malignancy. Here we show that oncogene-imposed dosage constraints can produce mutations that mimic tumor suppressor loss yet mark essential genes. In acute promyelocytic leukemia (APL), the transcription factor EBF3 gene harbors recurrent heterozygous loss-of-function mutations, a tumor suppressor signature, and is essential for APL survival. We resolve this paradox by showing that while PML::RARA, the fusion oncoprotein defining APL, drives EBF3 expression, excess EBF3 suppresses proliferation. Patient mutations reduce EBF3 activity through heterozygous inactivation, optimizing its expression within a corridor bounded by fitness constraints at both extremes. PML::RARA activates EBF3 via a p300-dependent intronic enhancer, and p300 inhibition exploits this constraint by suppressing EBF3 below the viability threshold. Our findings reveal that oncogenes can impose dosage constraints that drive adaptive mutations conventionally misclassified as tumor suppressor loss, with implications for interpreting cancer genomes.

cancer biology↗

bioRxiv2026

RNF185 destabilizes specific membrane proteins that span all ERAD branches

The endoplasmic reticulum (ER) is the primary site of eukaryotic membrane protein synthesis and quality control, which largely relies on ER-associated degradation (ERAD) to eliminate aberrant nascent proteins. In yeast, two ubiquitin ligases target proteins for ERAD based on aberrancies in substrate transmembrane (ERAD-M), lumenal (ERAD-L), or cytosolic (ERAD-C) domains. How an expanded repertoire of mammalian ERAD factors selects substrates across these classifications is unclear. Here, we show that the human ER-resident RNF185 ubiquitin ligase complex destabilizes a small but specific set of membrane proteins that span all three ERAD branches. Comparisons of three single-pass membrane proteins destabilized by RNF185 identify ERAD-M features in misoriented CHST10, ERAD-C features in unassembled SRPRB, and N-linked glycosylation-dependent ERAD-L features in ATP1B2. Our findings identify RNF185-destabilized membrane proteins with distinct aberrancies that collectively encompass all canonical ERAD substrate classifications and unexpectedly diverse quality control defects.

cell biology↗

bioRxiv2026

Traffickome infers directed, compartment-resolved membrane trafficking from proteomic data

Membrane trafficking determines the subcellular localization and compartment-residence time of receptors, channels, transporters and other transmembrane proteins and thereby influences where in the cell and how long these proteins function. Functional-enrichment computational approaches may predict the membrane-trafficking regulations but do not resolve the directionality of the transport between cellular compartments. We present Traffickome, a deterministic method organizing 754 trafficking proteins into 11 directed transitions across seven compartments and 1,837 signaling proteins across 22 pathways. Implemented as an interactive browser and open-source Python package, Traffickome infers compartment-to-compartment transport, cargo fate and signaling-pathway activity and enables in silico perturbation from a single proteomic comparison. Across 29 proteomic comparisons spanning 14 studies encompassing over 66,000 gene-level observations across over 10,000 genes, Traffickome ranked the expected trafficking transition first in 23 cases (79%), outperforming the best conventional enrichment methods (17 of 29, 59%). Mean accuracy fell to 18% after randomizing the protein-to-transition assignments. To illustrate the power of Traffickome, we performed time-resolved analysis of the phosphoproteome in EGF-stimulated cells with the goal to distinguish signaling by internalized versus plasma-membrane EGFR. In the resulting datasets, comprising over 18,000 phosphosite-level measurements, Traffickome revealed localization-dependent differences in EGF-induced phosphorylation and prioritized trafficking regulators for testing their effects on signaling processes.

cell biology↗

bioRxiv2026

Activation and inactivation pathways of a p53-like transcription factor govern lipid homeostasis in yeast

Membrane fluidity depends on unsaturated acyl chains that are generated in Saccharomyces cerevisiae by the desaturase Ole1, whose expression is primarily under the control of the transcription factor Mga2. Here, we show that the endoplasmic reticulum-anchored Mga2 precursor is ubiquitinated by the E3 ligase Rsp5 and then processively degraded by the proteasome until proteolysis stalls at a defined site, releasing a soluble fragment that enters the nucleus and activates Ole1 transcription. Unexpectedly, Mga2 contains a DNA-binding domain and a trans-activation-like segment structurally and functionally related to those of the human tumor suppressor p53. The mature transcription factor is degraded in the nucleus in a DNA binding-dependent manner; blocking this degradation causes unsaturated acyl chains to accumulate in lipid droplets, a detoxification response required for cell viability. These findings define the pathways that activate and inactivate Mga2, and uncover an evolutionary connection between the yeast lipid homeostasis regulator Mga2 and p53.

cell biology↗

bioRxiv2026

2-Hydroxyglutarate Redirects Fatty Acid Partitioning to Mitigate Lipotoxic Stress and Preserve Metabolic Fuel

The role of 2-hydroxyglutarate in lipid metabolism is currently unknown. Here we show that 2HG redistributes the partitioning of fatty acids into triglyceride storage and away from membrane phospholipid synthesis, mitochondrial oxidation, and lipotoxic intermediates. In primary human cardiac and vascular cells, both enantiomers, D2HG and L2HG, expanded triglyceride stores and lipid droplets while selectively depleting phosphatidylethanolamine, with L2HG acting more potently than D2HG despite lower intracellular accumulation. Mechanistically, L2HG increases DGAT-dependent triglyceride synthesis, slows triglyceride turnover, and constrains the ethanolamine branch of the Kennedy pathway. This response limits fatty acid oxidation, long-chain acylcarnitine accumulation, and lipid peroxidation independently of pseudohypoxic transcription or canonical lipid storage regulators, while also remodeling the phosphoproteome and redox proteome. L2HG accumulation induces hypertriglyceridemia in mice, redistributes the acyl chain composition of cardiac triglycerides, and limits ischemia-induced acylcarnitine accumulation in the heart, mirroring a positive association between circulating 2HG and triglycerides in humans. Thus, 2HG expands metabolic flexibility from whether fatty acids are used as fuel to how that fuel is allocated among storage, membrane synthesis, and oxidation.

cell biology↗

bioRxiv2026

Structural Mechanisms of DNAJC13 Dimeric Assembly and InsP6 binding in Recycling Endosome Regulation

The balance of plasma membrane protein degradation and recycling during endocytosis is regulated, in part, by the large J-domain-containing protein DNAJC13/RME-8 and WASH complexes, which function together with Retromer to support cargo trafficking into recycling endosomes. Despite extensive cellular and biochemical studies, the structural basis and proteomic landscape of DNAJC13 function remain elusive. Here, we find that DNAJC13 forms an unexpected antiparallel homodimeric architecture involving distinctive symmetrical interactions between composite IWN1 and -solenoid ARM2 domains in each protomer. Additionally, two PH-like domains of unknown function (PHL2 and PHL3), adjacent to the PI(3)P-binding PHL1 domain, form an unanticipated composite, positively charged pocket occupied by InsP6, as visualized structurally and verified by mass spectrometry. Proteomic profiling of DNAJC13-associated endosomes revealed enrichment of recycling endosomal components and WASH complexes. Mutations disrupting the dimer interface disable recruitment of WASH complexes to endosomes and result in elongated endosomal tubulation. Disruption of InsP6 binding impairs DNAJC13 binding to PI(3)P-containing vesicles in vitro and Transferrin-positive endosomes in cells. We further demonstrate that DNAJC13 dimerization and InsP6 binding promote melanin production during melanosome maturation, a process known to require recycling endosomes. This work provides a structural and mechanistic framework for understanding DNAJC13 function in recycling endosome control.

cell biology↗

bioRxiv2026

Activity-dependent lipid droplet biogenesis and turnover regulate synaptic integrity

Lipid droplets (LDs) are conserved organelles that buffer lipid storage and stress, yet their dynamics and functions in neurons remain largely unknown. Here, we report activity-dependent dynamics of neuronal LDs, visualized by a novel, genetically encoded LD reporter (termed LipiDew), in both cultured neurons and mouse motor cortex. Using LipiDew, we found that various paradigms of neuronal activation induced predominant and transient formation of LDs in neurites. Disruption of autophagic LD degradation (lipophagy) resulted in abnormal lipid accumulation in dendritic spines and shafts, promoted recruitment of synaptic scaffolding proteins to LDs, and altered intracellular calcium kinetics in neurons. In addition, mice with neuron-specific genetic impairment of lipophagy showed motor function defects. Together, these findings identify activity-dependent LD formation and lipophagic clearance in neuronal compartments as a crucial regulatory mechanism of synaptic integrity and neuronal function.

neuroscience↗

bioRxiv2026

Amino Acid Stress Induces Non-AUG Initiation of c-myc Translation

Initiation of translation at non-AUG start codons can generate novel isoforms of important cellular regulators, with activities or localization distinct from their AUG-initiated counterparts. Ribosomes scanning the 5UTR upstream of canonical AUG starts recognize near-cognate starts inefficiently, however, and the mechanisms by which non-canonical starts might be regulated physiologically are poorly understood. We show here that the restriction of utilization of individual amino acid induces a switch in expression of c-myc from its AUG-initiated isoform, c-myc-2, to an upstream CUG-initiated isoform, c-myc-1. This switch is enhanced in cells lacking GCN2, the protein kinase linking amino acid stress to phosphorylation and inhibition of eif2. Induction of upstream CUG start site usage in c-myc by restriction of proline utilization depends on the proximity of proline codons downstream of the canonical AUG start site. These observations support a model in which the stalling of an elongating ribosome near a canonical start leads to the accumulation of a ribosome queue that facilitates initiation at an upstream non-canonical start site. We find similar effects associated with deprivation of amino acids other than proline, and isoform changes with ATF-3 as well as c-myc. These findings suggest a broad mechanism by which amino acid stress can regulate translation initiation site use to modify the N-terminal proteome.

cell biology↗

bioRxiv2026

Regulation of the proteasome 20S core particle by the Parkinsonism-associated Proteins FBXO7 and PI31

FBXO7 and PI31 variants are linked to rare Parkinsonian syndromes, implicating their dysfunction in neurodegeneration. We define how both engage each other and regulate the proteasome 20S core particle (CP). In cells, each can associate independently with the proteasome, with multiple domains in FBXO7 contributing. Utilizing cryo-EM we visualized how FBXO7s C-terminal domain engages multiple subunits within the CP interior, blocking the {beta}5 peptidase activity. In contrast, we visualized PI31 engagement of all three catalytic sites within the 20S CP, revealing the previously unknown structural basis for {beta}1 inhibition. Furthermore, we establish how disease-associated variants impact both FBXO7 and PI31 function, including disruption of proteasome inhibition and SKP1-FBXO7-PI31 complex assembly. These results establish an unexpected function for FBXO7, providing a mechanistic basis for investigation of its role in proteasome regulation in Parkinsons disease.

biochemistry↗

bioRxiv2026

CDK/mTOR-dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent degradation

The C-terminal to LisH (CTLH) complex is a modular multi-subunit E3 ligase with diverse biological functions, yet how its overall ubiquitylation activity is tuned remains unclear. Here, we identify CDK- and mTOR-dependent phosphorylation of the cognate E2 enzyme UBE2H as a key regulator of CTLH E3 catalytic capacity. Phosphorylation of two N-terminal serine residues (S3/S5) reduces UBE2H charging with ubiquitin, thereby limiting the pool of active E2 available to CTLH. Mitotic CDK activity inactivates UBE2H during mitosis, whereas mTOR restrains UBE2H charging in interphase to couple CTLH-dependent ubiquitylation to nutrient status. Preventing this phosphorylation maintains UBE2H charging, enhances CTLH-mediated substrate degradation, promotes CTLH subunit turnover, and causes proliferation and mitotic defects. Using hyperactive UBE2H, we identify two additional CTLH substrates, the mitotic kinase NEK9 and Angio-associated migratory cell protein (AAMP) and define a DR-like C-degron recognized by the CTLH subunit MKLN1. These findings reveal how regulation of an E2 enzyme by cell cycle and nutrient signaling pathways dynamically shape CTLH activity.

cell biology↗

bioRxiv2026

High Resolution Multi-Pass Astral Analyzer Quantification Enables Highly Multiplexed 35-Plex Tandem Mass Tag Proteomics

Tandem mass tags (TMT) allow highly multiplexed and thus high-throughput, precisely quantitative proteomic analysis. Incorporation of additional deuterated reporter channels has near-doubled the multiplexation achieved with Thermo Scientific TMTpro reagents from 18 to 35-plex but requires extremely high [~]100k analyzer resolving power at m/z 128 to differentiate and quantify reporter ion channels, far beyond any single reflection time-of-flight analyzer, and exceeding the multi-reflection Thermo Scientific Astral analyzer in its standard operation. A multi-pass mode of Astral operation has been developed for the Thermo Scientific Orbitrap Astral Zoom mass spectrometer that triples the ion path to 90 m, more than doubling resolving power for a narrow m/z range. This "TMT HR mode" has been integrated into a new method of TMT proteomic analysis that splits regular MS2 analysis of labeled peptides into paired measurements comprising wide mass range scans for peptide identification, and TMT HR mode scans for reporter ion quantification. The method has been shown to accurately quantify 32-plex labeled HeLa protein lysate and provide far greater depth of analysis as state-of-the-art Orbitrap-only methods, while analysis of 11-plex labeled yeast showed no analytical depth sacrificed vs regular Orbitrap Astral TMT analysis. Further comparative measurements of a 2-cell line 35-plex sample demonstrated greater analytical depth, and similar quantitative precision, to "gold standard" Orbitrap MS3 methods.

biochemistry↗

bioRxiv2026

Isogenic cortical organoids enable precision targeting of APP variant-specific pathways in Alzheimer's disease

Alzheimers disease (AD) lacks disease-modifying therapies, in part due to the limitations of existing disease models, which have struggled to capture the early pathogenic events leading to neuronal degeneration. Unfortunately, recent therapies targeting hallmarks of AD have proven inefficient in humans, and it is thus necessary to identify alternative targets. Here, by generating an isogenic panel of hiPSC-derived cortical organoids carrying familial AD-associated APP variants or the protective A673T variant, we identified distinct, actionable pathogenic pathways specific to each variant. Proteomic analyses revealed variant-specific molecular disruptions: A673V organoids show impairments in proteostasis and cholesterol metabolism, whereas KM670/671NL organoids exhibit mitochondrial bioenergetic defects. These signatures overlapped with dysregulated proteins in post-mortem AD brains, demonstrating the reliability of our in vitro model. Importantly, targeted interventions restored neuronal survival in a variant-specific manner: overexpression of the master regulator of lysosomal biogenesis, TFEB, rescued A673V neurons, while ferroptosis inhibition selectively protected KM670/671NL neurons. Overall, our results indicate that differential treatments can be tailored based on distinct genetic backgrounds, supporting the development of precision medicine approaches in AD.

neuroscience↗

bioRxiv2026

Components of an ESCRT-independent nuclear envelope assembly pathway

In many cells, the nuclear envelope (NE) must be reassembled after mitosis and holes in the nuclear membrane must be "sealed." During NE assembly, the NE-specific adaptor, Cmp7, recruits/activates ESCRT-III proteins to mediate NE sealing. However, recent evidence suggests the presence of additional mechanisms. In a screen using the fission yeast, S. japonicus, we recently implicated the ESCRT adaptor, Alx1, and a conserved, but little studied protein, Vid27, in Cmp7-independent NE assembly. Here, we provide direct evidence that Alx1 functions in a Cmp7- and ESCRT-independent NE assembly pathway via positive regulation of Vid27. Consistent with a role in membrane remodeling, Vid27 localizes to sites of postmitotic NE sealing and is essential in S. japonicus. Alx1 and Vid27 interact and mutations disrupting the predicted interaction interface abolish Alx1s enhancement of Vid27 function at the NE. These findings define components of a new Cmp7- and ESCRT-independent NE assembly pathway, advancing our understanding of mechanisms that maintain the integrity of the nucleus.

cell biology↗

bioRxiv2025

miRISC inhibition causes mitotic defects and synergizes with genotoxic agents in cancers

Although individual microRNAs (miRNAs) can have tumorigenic or tumor-suppressive properties, their overall role in cancer remains controversial. Here, we show that cancer tissues and cell lines are characterized by preferential accumulation of the high-molecular-weight miRNA-induced silencing complex (HMWR), the functionally active form of the effector complex responsible for miRNA-mediated gene repression. Experimentally induced disassembly of the HMWR impairs the growth of human tumor xenografts and of autochthonous tumors in mouse models of human cancer in vivo. Furthermore, disassembly of the HMWR increases chromosome mis-segregation, which synergized with genotoxic agents to potentiate cancer cell vulnerability and improve therapeutic response. These findings suggest pharmacologic inhibition of HMWR as a novel anti-cancer strategy.

cancer biology↗

bioRxiv2025

Pre-clinical validation of a novel AAV-mediated gene therapy for KCNV2 retinopathy improves visual function in a mouse model and expression in patient organoids

Voltage-gated (Kv) potassium channels are critical for neuronal physiology, and their dysfunction can lead to serious consequences. For example, mutations in the silent modulatory Kv8.2 subunit are known to cause irreversible inherited blindness (KCNV2 retinopathy). This is a currently incurable condition that causes lifelong visual loss, reduced visual acuity, photoaversion, night blindness and abnormal colour vision, alongside a distinctive supernormal electrophysiological (ERG) retinal response to light. In this study, we demonstrate that AAV-mediated gene replacement therapy delivering a codon-optimised human KCNV2 gene subretinally into Kv8.2 knock-out mice significantly restores retinal function. Treated mice exhibited improved ERG responses and correct expression of KCNV2 and its encoded Kv8.2 protein in photoreceptors. Recovery of visually guided scotopic and photopic optomotor responses to wildtype levels was achieved at lower vector doses, highlighting dose-dependent efficacy. Furthermore, treatment of human retinal organoids derived from a KCNV2 patient iPSC line resulted in substantial Kv8.2 protein rescue. This work provides the first preclinical proof-of-concept for the safety and therapeutic potential of gene therapy for KCNV2 retinopathy, laying a strong foundation for future clinical trials.

neuroscience↗