Biology subjects

Campos, A. R.

Publications and source records attributed to Campos, A. R..

13 recordsLinked to original sources

bioRxiv2025

Improving Recombinant Antibody Production Using FcBAR: An In Situ Approach to Detect and Amplify Protein-Protein Interactions

Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the in situ detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production in situ, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.

systems biology↗

bioRxiv2024

PTX3 Governs Fibroblast-Epithelial Dynamics in Lung Injury and Repair

Dysfunctional interactions between fibroblasts and epithelial cells contribute to the progression of chronic lung diseases, including idiopathic pulmonary fibrosis (IPF). In this study, we utilized a coculture model of human small airway epithelial cells and fibroblasts to investigate intercellular communication during disease progression. Our transcriptomic and proteomic profiling reveal that fibroblasts repair epithelial cells in acute injury by boosting epithelial fatty acid metabolism; conversely, they exacerbate epithelial damage in chronic injury scenarios. By delineating regulators involved in these responses, we identified pentraxin 3 (PTX3) as a key antifibrotic factor secreted by fibroblasts in response to acute epithelial injury. Importantly, PTX3 levels are decreased in bronchoalveolar lavage (BAL) samples from IPF patients compared to non-fibrotic controls, indicating a potential link between diminished PTX3 levels and fibrosis progression. Furthermore, adding PTX3 to chronically injured epithelial-fibroblast cocultures mitigated the pro-fibrotic response and restored the epithelial barrier integrity. These findings highlight the dual roles of fibroblasts and the critical function of PTX3 in lung injury and repair, offering insights for therapeutic strategies.

molecular biology↗

bioRxiv2024

Deep, unbiased and quantitative mass spectrometry-based plasma proteome analysis of individual responses to mRNA COVID-19 vaccine

Global campaign against COVID-19 have vaccinated a significant portion of the world population in recent years. Combating the COVID-19 pandemic with mRNA vaccines played a pivotal role in the global immunization effort. However, individual responses to a vaccine are diverse and lead to varying vaccination efficacy. Despite significant progress, a complete understanding of the molecular mechanisms driving the individual immune response to the COVID-19 vaccine remains elusive. To address this gap, we combined a novel nanoparticle-based proteomic workflow with tandem mass tag (TMT) labeling, to quantitatively assess the proteomic changes in a cohort of 12 volunteers following two doses of the Pfizer-BioNTech mRNA COVID-19 vaccine. This optimized protocol seamlessly integrates comprehensive proteome analysis with enhanced throughput by leveraging the enrichment of low-abundant plasma proteins by engineered nanoparticles. Our data demonstrate the ability of this nanoparticle-based workflow to quantify over 3,000 proteins from 48 human plasma samples, providing the deepest view into COVID-19 vaccine-related plasma proteome study. We identified 69 proteins exhibiting a boosted response to the vaccine after the second dose. Additionally, 74 proteins were differentially regulated between seven volunteers, who contracted COVID-19 despite receiving two doses of the vaccine, and the ones who did not contract COVID-19. These findings offer valuable insights into individual variability in response to vaccination, demonstrating the potential of personalized medicine approaches in vaccine development.

immunology↗

bioRxiv2023

Longitudinal Deep Multi-Omics Profiling in a CLN3Δex7/8 Minipig Model Reveals Novel Biomarker Signatures for Batten Disease

Development of therapies for CLN3 Batten disease, a rare pediatric lysosomal storage disorder, has been hindered by the lack of etiological insights and translatable biomarkers to clinics. Here, we used a deep multi-omics approach to discover new biomarkers using longitudinal serum samples from a porcine model of CLN3 disease. Comprehensive metabolomics was combined with a nanoparticle-based LC-MS-based proteomic profiling coupled with TMTpro 18-plex to generate quantitative data on 769 metabolites and 2,634 proteins, collectively the most exhaustive multi-omics profile conducted on serum from a porcine model, which was previously impossible due a to lack of efficient deep serum proteome profiling technologies compatible with model organisms. The presymptomatic disease state was characterized by elevations in glycerophosphodiester species and lysosomal proteases, while later timepoints were enriched with species involved in immune cell activation and sphingolipid metabolism. Cathepsin S, Cathepsin B, glycerophosphoinositol, and glycerophosphoethanolamine captured a large portion of the genotype-correlated variation between healthy and diseased animals, suggesting that an index score based on these analytes could have great utility in the clinic.

neuroscience↗

bioRxiv2023

Histone chaperone HIRA, Promyelocytic Leukemia (PML) protein and p62/SQSTM1 coordinate to regulate inflammation during cell senescence and aging.

Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory Senescence-Associated Secretory Phenotype (SASP), is a cause of aging. In senescent cells, Cytoplasmic Chromatin Fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. PML protein organizes PML nuclear bodies (NBs), also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRAs localization to PML NBs. Inactivation of HIRA does not directly block expression of NF-{kappa}B target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-{kappa}B pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in regulation of SASP.

cell biology↗

bioRxiv2022

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1 and HIF-2. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2 levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2 protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1 translation through unknown mechanisms. ISCA2 inhibition also triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF- levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2 and to induce ferroptosis in pVHL deficient cells.

cancer biology↗

bioRxiv2022

PSGL-1 directs early TCR signaling to repress metabolism and promote T cell exhaustion by modulating the TCF-1/TOX axis in CD8+ T cells.

Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

immunology↗

bioRxiv2021

Vascular proteome responses precede organ dysfunction in sepsis

Vascular dysfunction and organ failure are two distinct, albeit highly interconnected clinical outcomes linked to morbidity and mortality in human sepsis. The mechanisms driving vascular and parenchymal damage are dynamic and display significant molecular crosstalk between organs and tissues. Therefore, assessing their individual contribution to disease progression is technically challenging. Here, we hypothesize that dysregulated vascular responses predispose the organism to organ failure. To address this hypothesis, we have evaluated four major organs in a murine model of S. aureus sepsis by combining in vivo labeling of the endothelial proteome, data-independent acquisition (DIA) mass spectrometry, and an integrative computational pipeline. The data reveal, with unprecedented depth and throughput, that a septic insult evokes organ-specific proteome responses that are highly compartmentalized, synchronously coordinated, and significantly correlated with the progression of the disease. Vascular proteome changes were found to precede bacterial invasion and leukocyte infiltration into the organs, as well as to precede changes in various well-established cellular and biochemical correlates of systemic coagulopathy and tissue dysfunction. Importantly, our data suggests a potential role for the vascular proteome as a determinant of the susceptibility of the organs to undergo failure during sepsis.

immunology↗

bioRxiv2021

A BioID-derived proximity interactome for SARS-CoV-2 proteins

The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here we use BioID to map the SARS-CoV-2 virus-host interactome using human lung cancer derived A549 cells expressing individual SARS-CoV-2 viral proteins. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are in association with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Collectively, these studies provide a valuable resource to potentially uncover novel SARS-CoV-2 biology and inform development of antivirals.

molecular biology↗

bioRxiv2021

Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. AKT is a relevant target for inhibition, in addition to KIT, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated significant synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to cell cycle arrest and cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

cancer biology↗

bioRxiv2020

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. One-sentence summarySARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

bioinformatics↗

bioRxiv2020

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum

Factor VIII (FVIII) is the coagulation factor deficient in hemophilia A, which is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low level secretion. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here we identified intrinsic and extrinsic factors that cause FVIII to form aggregates in the ER. We show that FVIII forms amyloid-like fibrils within the ER upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperones and their co-chaperones, BiP and CANX/CRT, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed {beta}-sheet polymerization and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein folding in general and have implication for ongoing hemophilia A gene therapy clinical trials. Key Points- FVIII forms amyloid aggregates in the ER that are dissolved in a chaperone- and glucose-dependent manner to produce secreted active FVIII. - A short amino acid sequence in the A1 domain causes {beta} sheet polymerization and ER chaperone BiP binding to this site prevents aggregation.

molecular biology↗

bioRxiv2019

A reference map of the human proinsulin biosynthetic interaction network

The beta-cell protein synthetic machinery is dedicated to the production of insulin, which plays a critical role in organismal homeostasis. Insulin synthesis requires the proper folding and trafficking of its precursor, proinsulin, yet the precise network of proinsulin protein interactions in the secretory pathway remains poorly defined. In the present study we conducted unbiased profiling of the proinsulin interactome in human islets, utilizing a human proinsulin-specific monoclonal antibody for affinity purification and mass spectrometry. Stringent analysis identified a central node of interactions between human proinsulin and sequential secretory pathway proteins that is remarkably conserved across 3 ethnicities and both genders. Among the most prominent proinsulin interactions was with ER-localized peroxiredoxin-4 (PRDX4). A functional role for PRDX4 in beta-cells was demonstrated by gene silencing that rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress. Conversely, exogenous PRDX4 improved proinsulin folding. Notably, oxidative stress and even high glucose treatment alone induced proinsulin misfolding in human islets and MIN6 cells, and this was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. This finding prompted PRDX4 analysis in a panel of human islet samples that revealed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of healthy individuals. Taken together, these data highlight the importance of elucidating the complete proinsulin interactome in human islets in order to understand critical steps controlling insulin biosynthesis, beta cell function, and T2D.

biochemistry↗