Biology subjects

Verdejo-Torres, O.

Publications and source records attributed to Verdejo-Torres, O..

7 recordsLinked to original sources

bioRxiv2025

The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

SWI/SNF chromatin remodeling complexes regulate gene expression by modulating nucleosome positioning, yet their roles in metal homeostasis during skeletal muscle development remain unclear. Here, we uncover distinct functions of the BAF, PBAF, and ncBAF complexes in myoblast proliferation under metal stress. While knockdown (KD) of Baf250a (BAF-specific) or Brd9 (ncBAF-specific) reduces myoblast proliferation, Baf180 (PBAF-specific) KD does not impair cell proliferation under basal conditions. Interestingly, supplementation with copper (Cu) or zinc (Zn) rescues proliferation in Baf250a- and Brd9-deficient myoblasts but paradoxically inhibits growth in Baf180 KD cells. Mechanistically, Baf180 KD disrupts Cu and Zn homeostasis, leading to intracellular Cu accumulation without labile Cu pools and impaired expression of Atp7a, a key Cu exporter. Transcriptomic analyses reveal widespread gene dysregulation in metal-treated Baf180-deficient cells, while metal supplementation promotes pro-proliferative gene expression in Baf250a- and Brd9-KD myoblasts. CUT&RUN assays demonstrate that metal-responsive transcription factor Mtf1 exhibits increased chromatin binding upon Cu treatment, targeting genes involved in stress response and myogenesis. Notably, Mtf1 colocalizes with Baf180 in the nucleus and co-immunoprecipitates with both conserved SWI/SNF subunits and Baf180, suggesting a functional interplay between PBAF and Mtf1 in regulating metal-dependent gene expression. Our findings establish the PBAF complex as a crucial regulator of Cu/Zn homeostasis in myoblast proliferation via Mtf1, while metal supplementation compensates for BAF and ncBAF dysfunction but exacerbates defects in PBAF-deficient cells. This study reveals a novel link between chromatin remodeling, metal signaling, and muscle development, with implications for stress adaptation and metabolic regulation in myogenesis.

molecular biology↗

bioRxiv2024

Cysteine Rich Intestinal Protein 2 is a copper-Responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

molecular biology↗

bioRxiv2024

Muscle-Specific Pyruvate Kinase Isoforms, Pkm1 and Pkm2, Regulate Mammalian SWI/SNF Proteins and Histone 3 Phosphorylation During Myoblast Differentiation

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, Pkm1 and Pkm2, function in glycolysis, but Pkm2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of Pkm1 and Pkm2 during myoblast differentiation. RNA-seq analysis revealed that Pkm2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. Dpf2 and Baf250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for activation of myogenic gene expression during differentiation. Pkm2 also mediated the incorporation of Dpf2 and Baf250a into the regulatory sequences controlling myogenic gene expression. Pkm1 did not affect expression but was required for nuclear localization of Dpf2. Additionally, Pkm2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters, but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for Pkm2 and a novel function for Pkm1 in gene expression and chromatin regulation during myoblast differentiation.

molecular biology↗

bioRxiv2024

PRMT5 orchestrates EGFR and AKT networks to activate NFκB and promote EMT

Neuroblastoma remains a formidable challenge in pediatric oncology, representing 15% of cancer-related mortalities in children. Despite advancements in combinatorial and targeted treatments improving survival rates, nearly 50% of patients with high-risk neuroblastoma will ultimately succumb to their disease. Dysregulation of the epithelial-mesenchymal transition (EMT) is a key mechanism of tumor cell dissemination, resulting in metastasis and poor outcomes in many cancers. Our prior work identified PRMT5 as a key regulator of EMT via methylation of AKT at arginine 15, enhancing the expression of EMT-driving transcription factors and facilitating metastasis. Here, we identify that PRMT5 directly regulates the transcription of the epidermal growth factor receptor (EGFR). PRMT5, through independent modulation of the EGFR and AKT pathways, orchestrates the activation of NF{kappa}B, resulting in the upregulation of the pro-EMT transcription factors ZEB1, SNAIL, and TWIST1. Notably, EGFR and AKT form a compensatory feedback loop, reinforcing the expression of these EMT transcription factors. Small molecule inhibition of PRMT5 methyltransferase activity disrupts EGFR/AKT signaling, suppresses EMT transcription factor expression and ablates tumor growth in vivo. Our findings underscore the pivotal role of PRMT5 in the control of the EMT program in high-risk neuroblastoma.

cancer biology↗

bioRxiv2023

Single nucleotide polymorphisms and Zn transport by nuclear ZIP11 shape cancer phenotypes in HeLa cells

Zinc (Zn) is an essential micronutrient that regulates critical biological processes such as enzymatic function, gene expression, and cell signaling and provides structural stability to proteins. Under physiological conditions, Zn is a divalent cation (Zn2+) in an inactive redox state. Maintaining Zn homeostasis is essential for normal cell development and function, and any dysregulation in supply and transport can lead to pathophysiological conditions. Zn transporters, such as ZIP11, are critical regulators in Zn homeostasis. In mammals, ZIP11 belongs to the GufA subfamily of ZIP transporters and is primarily found in the nucleus and the Golgi apparatus. Our laboratory recently reported an essential role of ZIP11 in maintaining nuclear Zn levels in the cervical cancer cell line HeLa that supports various hallmark phenotypes of cancer. Genomic analysis of publicly available cervical and ovarian cancer patient datasets identified several single-nucleotide polymorphisms (SNPs) in the ZIP11 coding region that correlated with disease severity. We hypothesized that these SNPs might have potentially deleterious consequences as they are in coding regions that could affect ZIP11 function by increasing substrate accessibility, potentially enhancing the carcinogenic phenotype of HeLa cells. In addition, we identified a classic Zn-metal binding site (MBS) composed of three relevant residues which may be required for transmembrane Zn-transport, maintenance of metal homeostasis, and the carcinogenic properties of HeLa cells. To address these questions, we utilized our well-established model of stably knock down (KD) ZIP11 in HeLa cells and overexpressed ZIP11 encoding single mutations corresponding to the SNPs and the MBS. Overexpression of ZIP11 encoding SNPs restored the Zn levels and the proliferation, migration, and invasive defects of ZIP11 KD cells. On the other hand, cells expressing ZIP11 with single MBS mutations exhibited a phenotype similar to KD cells, suggesting that Zn transport by this transporter is necessary for establishing and maintaining carcinogenic properties. The work highlights the functional relevance of nuclear Zn transport by ZIP11 to maintain homeostasis and support carcinogenic properties in ovarian cancer cells.

cell biology↗

bioRxiv2022

Implications of the 375W mutation for HIV-1 tropism and vaccine development.

HIV-1 vaccines need to induce broadly neutralizing antibodies (bnAb) against conserved epitopes in the envelope glycoprotein (Env) to protect against diverse HIV-1 clades. To achieve this, we need to understand how different amino acids affect the Env trimer structure to find a common strategy to readily produce Env vaccines of different subtypes. Previously, using a saturation mutagenesis strategy we identified single Env substitutions that open the CD4bs without modifying the trimer apex. One of these substitutions was a tryptophan residue introduced at position 375. Here, we introduced 375W into a large panel of 27 T/F, acute stage, chronic infection, and AIDS M-tropic, and non-M-tropic primary isolates from clades A, B, C, D and G, and circulating recombinant forms (CRFs) (CRF02_AG, and CRF01_AE), and a complex (cpx) (CRF13_cpx). To understand the effect of 375W mutation on Env trimer structure and tropism, we evaluated soluble (sCD4) and monoclonal antibody (mAb) neutralization of wt and mutant Env+ pseudovirions using bnAbs (b6, 17b, b12, VCR01, 3BNC117, PGT128, 10-1074, PGT145, PG9 and PG16), as well as macrophage infection. Broadly neutralizing Abs (bnAbs) such VCR01, and 3BNC117 neutralized almost all the primary isolates tested while the other bnAbs neutralized many but not all of our panel. In general, 375W did not impair or abrogate neutralization of potent bnAbs. However, b12 and VCR01 showed some tendencies to neutralize 375W macrophage-tropic (mac-tropic) and intermediate mac-tropic mutants more efficiently compared with non-mac-tropic mutants. We identify wt and 375W mutant Envs in our panel that infected macrophages more efficiently than non-mac-tropic variants but did not reach the levels of highly macrophage-tropic brain reference Envs. These partial mac-tropic Envs were classified as intermediate mac-tropic variants. Surprisingly, we observed a mac-tropic (clade G) and intermediate mac-tropic (clade C, and D) primary isolates wt Envs that were not derived from the central nervous system (CNS). The 375W substitution increased sensitivity to sCD4 in all Envs of our panel and increased macrophage infection in many Envs tested including a CRF01_AE X4 variant. However, variants already highly mac-tropic were compromised indicating the presence of other factors implicated in mac-tropism. Increased sCD4 sensitivity and enhanced macrophage infection provide strong evidence that 375W confers exposure of the CD4bs across Envs from different clades/CRF/cpx and disease stages. Enhanced exposure of the CD4bs by 375W had little or no effect on exposure and sensitivity of CD4bs epitopes targeted by potent bnAbs. In summary, we show that 375W consistently increases Env binding to CD4 for diverse Envs from different clades and disease stages, 375W exposure of CD4 receptor is a biologically functional substitution that alone confers mac-tropism on non-mac-tropic Envs and 3) 375W is an ideal substitution for inclusion into HIV vaccines constructed from different subtype Envs, with the aim to elicit neutralizing antibodies that target the CD4bs while maintaining exposure of other Env broad neutralization sites, and 4) we found mac-tropic and intermediate mac-tropic Envs from blood indicating that these Envs could evolve outside of CNS or be released from Brain. SignificanceSubstitutions exposing the CD4 binding site (CD4bs) on HIV-1 trimers, but still occluding non-neutralizing, immunogenic epitopes are desirable to develop HIV-1 vaccines. If such substitutions induce similar structural changes in trimers across diverse clades, they could be exploited in development of multi-clade Envelope vaccines. We show the 375W substitution increases CD4 affinity for Envelopes of all clades, circulating recombinant forms and complex Envs tested, independent of disease stage. Clade B and C Envs with an exposed CD4bs were described for macrophage-tropic strains from central nervous system (CNS). Here, we show that intermediate (clade C, and D) and macrophage-tropic (clade G) Envelopes can be detected outside CNS. Vaccines targeting the CD4bs will be particularly effective against such strains and CNS disease.

microbiology↗

bioRxiv2020

Protein arginine methyltransferase 5 promotes metastasis via enhancing EGFR transcription and modulating AKT1 activation by methylation

Protein arginine methyltransferase 5 (PRMT5) regulates a wide range of physiological processes, including cancer cell proliferation and metastasis, by generating symmetric di-methyl-arginine marks on histones and non-histone proteins. Here, we report that PRMT5 directly regulates epidermal growth factor receptor (EGFR) transcription to control EGF stimulated EGFR signaling. Furthermore, PRMT5 modulates protein kinase B (AKT) activation by methylation of AKT1 Arg 15, which is required for its subsequent phosphorylation at AKT1 Thr 308 and Ser 473. The PRMT5/EGFR/AKT axis converges to regulate transcription factors ZEB1, SNAIL, and TWIST1 to promote the epithelial-mesenchymal transition (EMT), in the manner that EGFR and AKT1 compensate each other to support tumor cell invasion and metastasis. Inhibiting PRMT5 methyltransferase activity with a small molecule inhibitor attenuated primary tumor growth and prevented hepatic metastasis in aggressive in vivo tumor models. Collectively, our results support the use of PRMT5 based therapies for metastatic cancer.

cancer biology↗