Biology subjects

Padilla-Rodriguez, M.

Publications and source records attributed to Padilla-Rodriguez, M..

5 recordsLinked to original sources

bioRxiv2026

Ceramide Synthases Regulate Myristate-Induced Intestinal IRE1α Activation

Background & AimsHigh-fat diets (HFDs) are a major modifiable risk factor for intestinal health. Current research focuses primarily on palmitate (C16:0); however, myristate (C14:0, rich in dairy products) has been minimally investigated. HFDs increase ceramide generation which drives endoplasmic reticulum (ER) stress; with both sphingolipids and ER stress being key contributors to intestinal biology. Whether different fatty acids uniquely impact sphingolipid metabolism and ER stress in intestinal biology has not been well defined. MethodsHuman colon epithelial cells were utilized to determine the role of ceramide synthases (CerS) 5 and 6 on myristate-induced ER stress using pharmacologic inhibitors and siRNA. Intestinal epithelial cell specific CerS5 and/or CerS6 knockout mice of both sexes were fed a control, high milk-fat, or high lard-fat diet for 16 weeks. Cells and colon tissues were analyzed for lipids, mRNA, and protein. ResultsMyristate treatment increased C14:0-ceramide and induced IRE1-dependent ER stress. Inhibition of CerS suppressed these effects, yet knockdown of CerS5/6, the primary enzymes generating C14:0-ceramide, unexpectedly exacerbated IRE1 activation both in vitro and in vivo, potentially due to depletion of dihydro(dh)sphingosine. ConclusionsCerS are required for myristate-induced IRE1 activation and restoration of the sphingoid base pool provides partial protection from intestinal ER stress. SYNOPSISThis study identifies a new mechanism linking dietary fats to intestinal cell stress. Ceramide synthases drive ER stress triggered by myristate, a dairy-derived fat, while restoring sphingoid bases partially protects cells, revealing a new role for sphingolipids in shaping intestinal responses to diet. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/728542v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@d66503org.highwire.dtl.DTLVardef@181bffaorg.highwire.dtl.DTLVardef@162f675org.highwire.dtl.DTLVardef@22a511_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Global Profiling of Remodeled Subcellular Structures Due to Drug Treatment and Disease

Cellular biochemistry arises from various interactions between macromolecules, including proteins, nucleic acids, and lipids. These make up membrane-bound organelles, membrane-less compartments, and molecular assemblies and scaffolds. Changes due to stimuli or disease can significantly impact cell fate and metabolism. We recently reported our protocol combining crosslinking and size exclusion chromatography with mass spectrometry (SEC-MS). In this study, we explore global changes to subcellular structure in Ewing sarcoma cell lines or in response to drug treatment. Crosslinking-dependent differential enrichment across four human cell lines is seen for proteins involved in splicing, mitochondria function, and cell division. We confirmed changes to nucleoli structure. We also examine structures affected by a transcription inhibitor, flavopiridol. Following flavopiridol treatment, we observed changes to the levels of transcription and mRNA processing machinery present in large subcellular structures. Unexpected effects were also found, including structural changes to a cytoplasmic organelle, the peroxisome. Along with a reduction in peroxisome function, dissociation of peroxisome pore proteins PEX13 and PEX14 was detected by STORM microscopy. We conclude that SEC-MS combined with crosslinking is a valuable method to detect and quantify drug or disease effects on subcellular structures and may shed light on new aspects to mechanisms underlying their biologic outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/672480v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f01b55org.highwire.dtl.DTLVardef@9279d4org.highwire.dtl.DTLVardef@7c7492org.highwire.dtl.DTLVardef@c92ff4_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LICrosslinking plus SEC-MS shows global effects on subcellular structure in disease and treatment. C_LIO_LIEwing sarcoma cell lines tested present distinct molecular composition in splicing, mitochondria, and nucleoli. C_LIO_LIA transcription inhibitor flavopiridol disrupts peroxisome function and protein import pores C_LI

biochemistry↗

bioRxiv2025

Spatial Biology and Organoid Technologies Reveal a Potential Therapy-Resistant Cancer Stem Cell Population in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with a 5-year survival rate of less than 10%. Chemotherapy is the current standard-of-care (SOC) for advanced PDAC; however, treatment resistance driven by a complex and immunosuppressive tumor microenvironment (TME) limits its effectiveness. To address mechanisms of resistance, we studied cabozantinib (cabo), a multi-kinase inhibitor approved for several solid tumors. Cabo has shown promise in preclinical PDAC models and clinical trials, particularly when combined with immunotherapy, but its mechanisms of action within the human PDAC TME and its potential to overcome therapy resistance remain unclear. Using CosMx Spatial Molecular Imaging (CosMx SMI) and Orion Multiplex Immunofluorescence (MxIF), we analyzed PDAC tissues collected after first line systemic chemotherapy and the Whipple surgical procedure, as well as from liver or lung metastatic sites from patients with PDAC. To functionally model the TME, we established matched patient-derived organoid (PDO) co-cultures harboring cancer associated fibroblasts (CAFs), and autologous immune cells (IMM) (PDO/CAF/IMM). These models were used to evaluate the effects of cabo and pembrolizumab, an anti-PD1 immune checkpoint inhibitor, with benchmarking of findings to the patients TME. Spatial analysis of post-chemotherapy and metastatic PDAC tissues revealed heterogeneous cellular neighborhoods within the TME, including enrichment of Schwann cells, CAFs, T regulatory cells, and cancer stem cells (CSCs). Distinct niches were observed in metastatic liver tissues, characterized by mesenchymal stem cells, fibroblasts, including myCAFs and iCAFs, and CSCs expressing CD44 and TROP2. In PDO/CAF/IMM co-cultures, treatment with cabo in combination with pembrolizumab enhanced cancer cell death by depleting myeloid-derived suppressor cells (MDSCs) and promoted cytotoxic T lymphocyte proliferation. Across both patient tissues and treated co-cultures, a persistent SOC-resistant cancer stem cell population emerged that expressed CD44 variant 9 (CD44v9). Taken together, these integrative spatial and organoid-based studies demonstrate that cabo can remodel the PDAC TME and potentiate PD-1 immunotherapy in preclinical models, while resistance associates with a CD44v9+ CSC population, revealing a potential therapeutic target.

cancer biology↗

bioRxiv2025

Neuronal endolysosomal acidification relies on interactions between transmembrane protein 184B (TMEM184B) and the vesicular proton pump

Disruption of endolysosomal acidification is a hallmark of several neurodevelopmental and neurodegenerative disorders. Impaired acidification causes accumulation of toxic protein aggregates and disrupts neuronal homeostasis, yet the molecular mechanisms regulating endolysosomal pH in neurons remain poorly understood. A critical regulator of lumenal acidification is the vacuolar ATPase (V-ATPase), a proton pump whose activity depends on dynamic assembly of its V0 and V1 subdomains. In this study, we identify transmembrane protein 184B (TMEM184B) as a novel regulator of endolysosomal acidification in neurons. TMEM184B is an evolutionarily conserved 7-pass transmembrane protein required for synaptic structure and function, and sequence variation in TMEM184B causes neurodevelopmental disorders, but the mechanism for this effect is unknown. We performed proteomic analysis of TMEM184B-interacting proteins and identified enrichment of components involved in endosomal trafficking and function, including the V-ATPase. TMEM184B localizes to early and late endosomes, further supporting a role in the endosomal system. Loss of TMEM184B results in significant reductions in endolysosomal acidification within cultured mouse cortical neurons. This alteration in pH is associated with impaired assembly of the V-ATPase V0 and V1 subcomplexes in the TMEM184B mutant mouse brain, suggesting a mechanism by which TMEM184B promotes flux through the endosomal pathway. Overall, these findings identify a new contributor in maintaining endosomal function and provide a mechanistic basis for disrupted neuronal function in human TMEM184B-associated nervous system disorders. Significance StatementEndolysosomal acidification is essential for neuronal protein homeostasis, yet its regulation in neurons remains poorly understood. Here, we identify TMEM184B as a key regulator of this process, establishing its first known cellular role. We show that TMEM184B interacts with vacuolar ATPase (V-ATPase) components and promotes the assembly of its V0 and V1 subdomains, facilitating lumenal acidification. Loss of TMEM184B disrupts endolysosomal pH in neurons, potentially impairing proteostasis. These findings reveal a critical function for TMEM184B in neuronal maintenance and provide mechanistic insight into its link to neurological disorders. This work advances our understanding of endolysosomal regulation and suggests TMEM184B regulation could improve outcomes in diseases involving lysosomal dysfunction.

neuroscience↗

bioRxiv2025

Complex roles for proliferating cell nuclear antigen in human cytomegalovirus replication

DNA viruses at once elicit and commandeer host pathways, including DNA repair pathways for virus replication. Despite encoding its own DNA polymerase and processivity factor, human cytomegalovirus (HCMV) recruits the cellular processivity factor, proliferating cell nuclear antigen (PCNA) and specialized host DNA polymerases involved in translesion synthesis (TLS) to replication compartments (RCs) where viral DNA (vDNA) is synthesized. While the recruitment of TLS polymerases is important for viral genome stability, the role of PCNA is poorly understood. PCNA function in DNA repair is regulated by monoubiquitination (mUb) or SUMOylation of PCNA at lysine 164 (K164). We find that mUb-PCNA increases over the course of infection, and modification of K164 is required for PCNA-mediated restriction of virus replication. mUb-PCNA plays important known roles in recruiting TLS polymerases to DNA, which we have shown are important for viral genome integrity and diversity, represented by novel junctions and single nucleotide variants (SNVs), respectively. We find that PCNA drives SNVs on vDNA similar to Y-family TLS polymerases, but that this did not require modification at K164. Unlike TLS polymerases, PCNA was dispensable for preventing large scale rearrangements on vDNA. These striking results suggest separable PCNA-dependent and - independent functions of TLS polymerases on vDNA. By extension, these results imply roles for TLS polymerase beyond their canonical function in TLS in host biology. These findings highlight PCNA as a complex restriction factor for HCMV infection, likely with multiple distinct roles, and provides new insights into the PCNA-mediated regulation of DNA synthesis and repair in viral infection. IMPORTANCEGenome synthesis is a critical step of virus life cycles and a major target of antiviral drugs. Human cytomegalovirus, like other herpesviruses, encodes machinery sufficient for viral DNA synthesis and relies on host factors for efficient replication. We have shown that host DNA repair factors play important roles in HCMV replication, but our understanding of this is incomplete. Building on previous findings that specialized host DNA polymerases contribute to HCMV genome integrity and diversity, we sought to determine the importance of PCNA, the central polymerase regulator. PCNA associates with nascent viral DNA and restricts HCMV replication. While PCNA is dispensable for genome integrity, it contributes to genome diversity. Our findings suggest that host polymerases function on viral genomes by separable PCNA-dependent and - independent mechanisms. Through revealing complex roles for PCNA in HCMV replication, this study expands the repertoire of host DNA synthesis and repair proteins hijacked by this ubiquitous herpesvirus.

microbiology↗