Biology subjects

PADILLA-BENAVIDES, T.

Publications and source records attributed to PADILLA-BENAVIDES, T..

11 recordsLinked to original sources

bioRxiv2026

CuiT is a Cu importer required for metal homeostasis in Salmonella enterica

Copper (Cu) is an essential micronutrient that serves as a cofactor for redox enzymes but becomes toxic when unregulated. In bacteria, while Cu efflux systems are well characterized, mechanisms of Cu import remain poorly understood. Here, we characterize the major facilitator superfamily transporter CuiT (STM1486) as a key Cu importer in Salmonella enterica. Comparative genomics revealed that cuiT is evolutionarily conserved across Enterobacteriaceae, and structural modeling predicts a 12-transmembrane-helix architecture with conserved His, Met, and Cys residues suitable for Cu coordination. Functional analyses demonstrated that deletion of cuiT reduces intracellular Cu accumulation, slows Cu uptake kinetics, and diminishes expression of Cu-responsive genes, including copA, cueP, cueO, and golB. Conversely, overexpression of CuiT increases intracellular Cu but sensitizes cells to Cu stress, highlighting the need for tight regulation. Kinetic modeling indicates that CuiT mediates rapid Cu import, supporting larger intracellular Cu pools compared to Pseudomonas influx transporters. These findings position CuiT as a central component of the Salmonella Cu homeostasis network, linking Cu import to transcriptional regulation, redox balance, and stress adaptation. Our work provides mechanistic insights into bacterial Cu acquisition and suggests CuiT and associated pathways as potential targets for antimicrobial strategies. SignificanceCopper (Cu) is essential for bacterial redox enzymes but toxic when dysregulated. While Cu efflux pathways are well studied, mechanisms of Cu import are poorly understood. We identify CuiT, a conserved major facilitator superfamily transporter, as a key Cu importer in Salmonella enterica. CuiT controls intracellular Cu levels, influences Cu-responsive gene expression, and maintains redox balance and stress adaptation. Disruption or overexpression of CuiT perturbs Cu homeostasis, highlighting its regulatory importance. These findings reveal a critical bacterial Cu acquisition pathway and suggest CuiT and its network as potential antimicrobial targets, advancing understanding of metal homeostasis in pathogens.

microbiology↗

bioRxiv2026

Reciprocal regulation between the protein arginine deiminases and mSWI/SNF chromatin remodelers controls skeletal muscle differentiation and regeneration

Protein arginine deiminases (PADs) post-translationally convert arginine to citrulline on target proteins and serve as regulators of multiple cellular functions. PAD enzymes have been implicated in autoimmune disorders, cancer, and other diseases. Inhibiting PAD activity is currently being pursued clinically for therapeutic purposes. However, little is known about PAD function in normal developmental or homeostatic processes. Here we show that multiple PAD isoforms contribute to primary myoblast differentiation by binding to regulatory regions of target genes. Furthermore, we demonstrate a novel, reciprocal requirement for PADs and mammalian SWI/SNF (mSWI/SNF) chromatin remodeling enzymes; PAD enzymes are required for the expression and binding of specific mSWI/SNF enzyme subunits to target gene regulatory sequences while mSWI/SNF enzymes are required for the expression and binding of PAD enzymes. In vivo, the PADs contribute to mouse skeletal muscle regeneration after injury, with PAD4 specifically identified as a required regulator. This work identifies the PADs as critical cofactors in the initiation of skeletal muscle differentiation and reveals previously unappreciated connections between two major co-activator families during normal tissue development. Moreover, the results reveal important considerations for ongoing therapeutic approaches to myriad human diseases that utilize inhibitors of each enzyme family.

molecular biology↗

bioRxiv2026

Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

Flavonoids are naturally occurring polyphenolic compounds that have been extensively explored as scaffolds for drug development due to their diverse biological activities. Brazilin, a homoisoflavonoid with reported antitumoral properties, does not fully meet pharmaceutical criteria, and chemical modification of natural compounds is often required to enhance bioactivity and efficacy. Here, we evaluated the pro-apoptotic activity of Brazilin and its semi-synthesized methoxylated (OMe)3 and acetylated (OAc)3 derivatives in triple-negative MDA-MB-231 and luminal A MCF7 breast cancer cell lines. We assessed cell viability, proliferation, oxidative stress, and mitochondrial integrity, and analyzed apoptotic features using confocal microscopy, western blotting, and RT-qPCR. In addition, RNA sequencing was performed to characterize transcriptomic changes in MDA-MB-231 cells following treatment with unmodified Brazilin or its derivatives. Brazilin and Brazilin-(OAc)3 significantly reduced cell viability and proliferation in MDA-MB-231 cells, whereas MCF7 cells exhibited increased viability and growth in response to Brazilin-(OMe)3. In MDA-MB-231 cells, treatment with Brazilin and Brazilin-(OAc)3 induced apoptosis-associated features, including chromatin condensation, {gamma}H2AX accumulation, and PARP cleavage. These effects were accompanied by a modest increase in mitochondrial oxidative stress and loss of mitochondrial membrane potential. Notably, no cytotoxic or apoptotic features were detected in non-tumorigenic MCF10A cells. Transcriptomic analysis revealed that Brazilin treatment upregulated genes associated with endoplasmic reticulum stress, including ATF3, in MDA-MB-231 cells. Collectively, our results indicate that Brazilin and its acetylated derivative selectively induce mitochondrial stress and cell death in triple-negative breast cancer cells, potentially involving ER stress pathways.

cancer biology↗

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↗

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↗

bioRxiv2023

Differential requirements for different subfamilies of the mammalian SWI/SNF chromatin remodeling enzymes in myoblast differentiation

Mammalian SWI/SNF (mSWI/SNF) complexes are ATP-dependent chromatin remodeling enzymes that are critical for normal cellular functions and that are mis-regulated in [~]20% of human cancers. These enzymes exhibit significant diversity in the composition of individual enzyme complexes. mSWI/SNF enzymes are classified into three general sub-families based on the presence or absence of specific subunit proteins. The three sub-families are called BAF (BRM or BRG1-associated factors), ncBAF (non-canonical BAF), and PBAF (Polybromo-associated BAF). The biological roles for the different subfamilies of mSWI/SNF enzymes are poorly described. We knocked down (KD) the expression of genes encoding subunit proteins unique to each of the three subfamilies, Baf250A, Brd9, and Baf180, which mark the BAF, ncBAF, and PBAF sub-families, respectively, and examined the requirement for each in myoblast differentiation. We found that BAF250A and the BAF complex was required to drive lineage-specific gene expression during myoblast differentiation. KD of Baf250A reduced the expression of the lineage determinant Myogenin and other differentiation markers, due to decreased binding of BAF250A to myogenic gene promoters. KD of Brd9 delayed myoblast differentiation. However, RNA-seq analysis revealed that while the Baf250A-dependent gene expression profile included genes involved in myogenesis, the Brd9-dependent gene expression profile did not. Moreover, no-colocalization of Baf250A and Brd9 was observed in differentiating cells, suggesting independent mechanisms of action for BAF and ncBAF complexes in myogenesis. The PBAF complex was dispensable for myoblast differentiation. The results distinguish between the roles of the mSWI/SNF enzyme subfamilies during myoblast differentiation.

molecular biology↗

bioRxiv2022

The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed with mitochondrial cuproenzymes, such as Sco1, Sco2 and Cox17, which contributes to the metallation of the oxidase. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, is also able to bind Cu+ and acts as an active copper transporter in eukaryotic cells through these membranes, and ultimately aid in the metallation of COX. We used a well-established differentiation model of primary myoblasts derived from mouse satellite cells, where Cu availability is necessary for growth and maturation, and showed PiC2 is a target of MTF1, its expression is induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were ameliorated by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also strongly suggest that PiC2 may act to directly load Cu into COX, which was verified by in vitro Cu+-transfer experiments. The data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria, COX and, subsequently, the differentiation of primary myoblasts.

cell biology↗

bioRxiv2019

Leptin induces cell migration and invasion in a FAK-Src- dependent manner in breast cancer cells

Breast cancer is the most common invasive neoplasia, and the second leading cause of death associated with cancer in women worldwide. Mammary tumorigenesis is severely linked to obesity, the potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloproteases secretion, and invasion. By Western blot we found that leptin activates FAK and Src, and induces the localization of FAK to the focal adhesions. Specific inhibitors of FAK and Src showed that the effect exerted by leptin in cell migration, and invasion in breast cancer cells is dependent on these kinases. Moreover, by gelatin zymmography we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9, in a FAK and Src dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

cell biology↗

bioRxiv2019

MTF1, a classic metal sensing transcription factor, promotes myogenesis in response to copper.

MTF1 is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements (MREs). MTF1 responds to metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. We used multiple strategies to identify an unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, MTF1 expression increased, as did nuclear localization. Mtf1 knockdown impaired differentiation, while addition of non-toxic concentrations of Cu+ enhanced MTF1 expression and promoted myogenesis. Cu+ bound stoichiometrically to a C-terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes and binding was stimulated by copper. MTF1 formed a complex with MyoD at myogenic promoters, the master transcriptional regulator of the myogenic lineage. These studies establish novel mechanisms by which copper and MTF1 regulate gene expression in myoblast differentiation.

cell biology↗

bioRxiv2018

Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

Trace elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) are enzyme cofactors and second messengers in cell signaling. Trace elements are emerging as key regulators of differentiation and development of mammalian tissues including blood, brain, and skeletal muscle. We previously reported an influx of Cu and dynamic expression of various metal transporters during differentiation of skeletal muscle cells. Here, we demonstrate that during differentiation of skeletal myoblasts an increase of additional trace elements such as Mn, Fe and Zn occurs. Interestingly the Mn increase is concomitant with increased Mn-dependent SOD2 levels. To better understand the Mn import pathway in skeletal muscle cells, we probed the functional relevance of the closely related proteins ZIP8 and ZIP14, which are implicated in Zn, Mn, and Fe transport. Partial depletion of ZIP8 severely impaired growth of myoblasts and led to cell death under differentiation conditions, indicating that ZIP8-mediated metal transport is essential in skeletal muscle cells. Moreover, knockdown of Zip8 impaired activity of the Mn-dependent SOD2. Growth defects were partially rescued by Mn supplementation to the medium, suggesting additional functions for ZIP8 in the skeletal muscle lineage. Knockdown of Zip14, on the other hand, had only a mild effect on myotube size, consistent with a role for ZIP14 in muscle hypertrophy. This is the first report on the functional relevance of two members of the ZIP family of metal transporters in the skeletal muscle lineage, and further supports the paradigm that trace metal transporters are critical modulators of mammalian tissue development.

cell biology↗