Biology subjects

Fernandez-Rodriguez, R.

Publications and source records attributed to Fernandez-Rodriguez, R..

9 recordsLinked to original sources

bioRxiv2026

A single Omicron mutation reshapes ORF3a-driven host-cell remodelling

SARS-CoV-2 ORF3a remodels host membranes, but the structural basis and metabolic consequences of this process remain unclear. Here, we combine complementary imaging approaches to define ORF3a function at nanometric scale, identifying underlying mechanisms, and determining how Omicron variant rewire this activity. ORF3a from the ancestral Wuhan strain disrupts Golgi cisternae, drives the formation of ORF3a dense vesicles, remodels mitochondrial architecture, and promotes lipid droplet expansion. Multi-omics analyses further reveal selective triacylglycerol accumulation linked to DGAT1 upregulation, which we validate pharmacologically through DGAT1 inhibition. In contrast, Omicron ORF3a variant, despite carrying only the Thr223Ile substitution within the {beta}7-{beta}8 loop at the bottom of the cytosolic domain, induced a dramatic phenotypic shift: ORF3a localizes to multivesicular bodies, preserves Golgi architecture, and fails to induce lipid accumulation. All together, these results identify ORF3a as a regulator of membrane organization and lipid homeostasis, showing how minimal sequence variation rewires host-cell remodelling. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/742305v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@135032borg.highwire.dtl.DTLVardef@1633d4borg.highwire.dtl.DTLVardef@4d4115org.highwire.dtl.DTLVardef@1ebdf65_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

A Human Recombinant Antibody Against DRP1 Serine 616 Phosphorylation Enables Detection of BRAFV600E-Associated Mitochondrial Division in Cancer

Mitochondria are dynamic organelles that continuously undergo balanced cycles of fusion and division to meet cellular demands. Mitochondrial division is mediated by Dynamin-Related Protein 1 (DRP1), a cytosolic large GTPase whose phosphorylation at serine 616 (DRP1-S616) promotes its translocation to the outer mitochondrial membrane and organelle division. Dysregulated, mitochondrial division disrupts cellular homeostasis and contributes to disease pathogenesis, including cancer. Our prior work demonstrated that the oncogene-induced mitogen-activated protein kinase (MAPK) pathway constitutively phosphorylates DRP1 at serine 616 (DRP1-S616), which is essential to cellular transformation and correlates with oncogene status in patient tissues. Similarly, DRP1-S616 is subject to pharmacologic control by targeted therapies against oncogenic MAPK signaling. Building upon this foundation, we developed a human recombinant monoclonal antibody with high specificity for DRP1-S616, referred to as 3G11. Using diverse biochemical platforms, we demonstrate the robust utility of 3G11 to detect DRP1-S616 in melanoma cell extracts and isolated organelles. Immunofluorescence revealed that pharmacologic inhibition of oncogenic MAPK signaling reduces DRP1-S616 levels which correlates with mitochondrial hyperfusion; while immunohistochemistry showed that elevated DRP1-S616 expression in human tissues correlates with BRAFV600E disease. Together, these findings establish 3G11 as a specific, versatile, renewable, and cost-effective tool for studying mitochondrial division, with strong potential for clinical applications.

cancer biology↗

bioRxiv2025

Single-Cell Dissection of Immunometabolic Rewiring in the Porcine Ileum during Salmonella Typhimurium Infection

Salmonella Typhimurium is a major zoonotic pathogen, with pigs acting as important subclinical carriers. To explore the specific intestinal immune response at the cellular level, we used Single-cell RNA sequencing (scRNA-seq), enabling detailed analysis of immune cell types and gene expression profiles during infection. In addition to enterocytes, our results revealed the presence of diverse immune populations, including monocytes/macrophages, dendritic cells, innate lymphoid cells (ILCs), thirteen T cell subtypes and five B cell populations were identified, revealing pronounced infection-driven alterations in cellular composition and transcriptional states. Among T cells, naive and follicular CD4+/CD8+ {beta} T cells and NK T cells were expanded, whereas effector CD8+ T cells and CD2- and SELLhi {gamma}{delta} T cells were decreased. B-cell populations shifted toward activated and cycling states, with decreased antibody-secreting, resting, and transitioning cells. Dendritic cells and monocyte/macrophage populations were expanded, and group 3 ILCs and enterocytes were markedly reduced. Pathway analyses revealed robust cell type-specific immunometabolic remodeling, including enhanced protein-folding and stress-adaptive pathways in T and B cells, heightened inflammatory, interferon, and cytokine signaling in myeloid populations, and coordinated metabolic and immune adjustments in epithelial cells, highlighting the complexity of host responses to Salmonella infection. This study provides the first scRNA-seq landscape of the porcine ileum during S. Typhimurium infection, offering insight into host immune cell dynamics and immunometabolic responses.

genomics↗

bioRxiv2025

SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition

SARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.

immunology↗

bioRxiv2025

Lymphangiogenesis driven by VEGF-C reshapes tumor immune landscape and enables tumor eradication by viral immunotherapy

Vascular Endothelial Growth Factor C (VEGF-C) is a key lymphangiogenic growth factor expressed by many types of cancer. Here, we investigated the effects of tumor-derived VEGF-C on the therapeutic efficacy of oncolytic virotherapy with avian paramyxoviruses APMV-1 (NDV) and AMPV-4. Treatment of B16F10 tumors not expressing VEGF-C with either virus led to tumor growth delay, and complete responses were rare. In contrast, when tumors expressed VEGF-C, viral therapy led to complete remission and long-term immunological memory in most mice. Upon re-challenge, most mice remained tumor- and metastasis-free for over 18 months, indicating durable immunity. VEGF-C induced tumor lymphangiogenesis, which correlated with high CD8+ and CD4+ T-cell densities in proximity of lymphatic vessels. Spectral flow cytometry revealed distinct changes in the composition and activation of CD8+, CD4+ T cells and NK cells associated with complete remission. In responders, tumors were highly enriched in CD8+CD25+ PD-1+ effector T cells. Composition of T cells was altered in sentinel and in contralateral lymph nodes, indicating a systemic immune response. Taken together, these data demonstrate VEGF-C-induced changes in tumor immune landscape which are critical for achieving complete response and support combining VEGF-C with APMV virotherapy as a novel highly effective strategy for cancer treatment.

cancer biology↗

bioRxiv2024

HLA-E and NKG2A Mediate Resistance to M. bovis BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer

BackgroundBacillus Calmette-Guerin (BCG) is the standard of care treatment for high-risk non-muscle-invasive bladder cancer (NMIBC), yet many patients develop recurrent disease despite evidence of ongoing immune activation. We investigated mechanisms of immune escape in BCG-unresponsive tumors and evaluated the therapeutic potential of targeting the HLA-E/NKG2A axis. MethodsSingle-cell RNA sequencing, spatial immunophenotyping, proteomic profiling, and functional ex vivo assays were performed using tumors and urine samples from patients with BCG-naive and BCG-unresponsive NMIBC. ResultsBCG-unresponsive tumors were enriched for HLA-E-expressing malignant cells compared with BCG-naive tumors. Increased HLA-E expression was associated with enhanced IFN-{gamma} signaling and was induced by IFN-{gamma} stimulation in primary tumor cells and bladder cancer tumor lines. Spatial analyses demonstrated accumulation of NKG2A+ NK and CD8 T cells in proximity to HLA-Ehigh tumor cells, with increased NKG2A:HLA-E interactions in BCG-unresponsive tumors. Despite high expression of cytotoxic mediators, NKG2A+ effector cells displayed impaired degranulation. Blockade of NKG2A with monalizumab restored degranulation of and cytotoxicity by tumor-infiltrating lymphocytes in autologous tumor co-cultures. ConclusionsBCG-unresponsive NMIBC tumors are enriched for HLA-E-expressing tumor cells and NKG2A+ effector lymphocytes, with increased engagement of the HLA-E/NKG2A axis within the tumor microenvironment. These findings identify the HLA-E/NKG2A axis as a therapeutic vulnerability and provide a rationale for clinical evaluation of NKG2A blockade as a bladder-sparing strategy for patients with BCG-unresponsive disease.

cancer biology↗

bioRxiv2023

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention. One-Sentence SummaryMitochondria and metabolic alterations induced by SARS- CoV-2 accessory proteins ORF3a, ORF9b, ORF9c, ORF10 in pulmonary cells unravel new targets of intervention.

immunology↗

bioRxiv2022

Antiviral immune responses, cellular metabolism and adhesion are differentially modulated by SARS-CoV-2 ORF7a or ORF7b

SARS-CoV-2, the causative agent of the present COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome, and some have been implicated in facilitating infection and pathogenesis through their interaction with cellular components. Among these proteins, accessory protein ORF7a and ORF7b functions are poorly understood. In this study, A549 cells were transduced to express ORF7a and ORF7b, respectively, to explore more in depth the role of each accessory protein in the pathological manifestation leading to COVID-19. Bioinformatic analysis and integration of transcriptome results identified defined canonical pathways and functional groupings revealing that after expression of ORF7a or ORF7b, the lung cells are potentially altered to create conditions more favorable for SARS-CoV-2, by inhibiting the IFN-I response, increasing proinflammatory cytokines release, and altering cell metabolic activity and adhesion. Based on these results, it is reasonable to suggest that ORF7a and ORF7b could be targeted by new therapies or used as future biomarkers during this pandemic.

immunology↗

bioRxiv2022

NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer

PD-1/PD-L1-blockade immunotherapies have limited efficacy in the treatment of muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Here, we show that KLRC1 (NKG2A) expression associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in CD8Ahigh bladder tumors. The loss of antigen presentation is a common mechanism for tumor escape in bladder cancer. NKG2A+ CD8 T cells are able to circumvent HLA-ABC loss through TCR-independent cytotoxicity, which is partly mediated by DNAM-1. In bladder tumors, NKG2A is acquired on a subset of PD-1+ CD8 T cells, alongside stronger tissue-residency memory features, TCR-independent cytotoxicity and evidence of recent proliferation. HLA-E is low but variably expressed on bladder tumors. When expressed, NKG2A+ CD8 T cell anti-tumor responses to HLA-ABC-deficient tumors are inhibited and partly restored upon NKG2A blockade. Overall, our study identifies an alternative path for CD8 T cell exhaustion, that is mediated by NKG2A upregulation and TCR-independent cytotoxicity.

immunology↗