Biology subjects

Calderwood, S. K.

Publications and source records attributed to Calderwood, S. K..

10 recordsLinked to original sources

bioRxiv2025

Novel DNA endonuclease activity of human ERK1 protein

ERK1 is a key kinase in the mitogen-activated protein kinase pathway, which transduces extracellular signals into cellular responses. Following triggering of the pathway, cytosolic ERK1 is phosphorylated, translocates into the nucleus, and activates DNA-bound transcription factors and target genes. This study reports a novel, non-canonical role of human ERK1 as a DNA endonuclease. It catalyzes single-strand DNA breaks in sequence-unspecific yet, topology-sensitive manners. ERK1 catalysis requires magnesium ions and no ATP consumption. Comprehensive biochemical and in silico structural analyses indicate that a Mg2+ pocket, including a residue N171, which is allosterically modulated by the N-terminal domain, is important for DNA substrate binding and catalysis. Fluorescence imaging analysis suggests that ERK1 may interact with cytoplasmic DNAs in molecular condensates and destabilize them. Collectively, this study reveals an unprecedented role of ERK1 in nicking DNA molecules, which highlights the potential to regulate the fate of cytoplasmic DNAs.

molecular biology↗

bioRxiv2025

DNA topological regulation by topoisomerase IIbeta-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

Hypoxic stress responses are essential for cellular and organismal survival and drive gene regulation across diverse biological pathways, including cell cycle progression and energy metabolism. Here, we show that topoisomerase II{beta} (TOP2B) regulates DNA topology and transcription of hypoxia-inducible genes (HIGs) in a DNA-dependent protein kinase (DNA-PK)-dependent manner. Integrated cellular, biochemical, and genomic analyses reveal an antagonistic yet correlated relationship between TOP2B and DNA-PK activities. Under normoxic conditions, TOP2B associates with HIGs and represses transcription by suppressing DNA negative supercoiling formation and accessibility. Under hypoxic exposure, TOP2B dissociates from HIGs, while DNA-PK and HIF1 are recruited to activate HIGs. Notably, DNA-PK is required for the repressive function of TOP2B, as DNA-PK knockout abrogates TOP2B release and activity, resulting in elevated expression of a number of HIGs. Mechanistically, DNA-PK phosphorylates TOP2B at T1403, stimulating its catalytic activity to restrain DNA unwinding and accessibility, thereby suppressing HIG transcription. Collectively, these findings identify a novel role for TOP2B and DNA-PK-mediated regulation of TOP2B as key transcriptional regulators of HIG expression. We propose that phosphorylation-dependent modulation of TOP2B activity is coordinated with transcriptional states and determines DNA topology to regulate Pol II transcription.

molecular biology↗

bioRxiv2024

Jerusalem artichoke extracts regulate the gene expression of key enzymes involved in fatty acid biosynthesis

Jerusalem artichoke (JA) is a traditional remedy for alleviating symptoms of diabetes. In fact, the suppressive effects of JA on blood sugar have been reported in multiple studies since 1934. Recent studies have indicated that type II diabetes is often caused by insulin resistance rather than insulin reduction and that increased blood and interstitial fatty acid levels contribute to insulin resistance and the development of diabetes. However, whether JA affects lipogenesis has not been studied. Here, we elucidated the effects of JA on the expression of two key enzymes involved in fatty acid biosynthesis, fatty acid synthase (FASN) and acetyl-coA carboxylase (ACACA), using three immortalized human bone marrow, colon, and liver cell lines. Caffeine and ICRF193, a catalytic inhibitor of topoisomerase II (TOP2), were included as positive controls, and JA was extracted into water- or dimethyl sulfoxide-soluble components, termed H-JA and D-JA. D-JA significantly reduced the expression of FASN and ACACA at the mRNA and protein levels. D-JA-treated cells exhibited altered TOP2 levels and FASN/ACACA expression appeared to be controlled by TOP2 activity and levels. Taken together, our study revealed a novel effect of JA extracts on inhibiting the expression of the key enzymes involved in the fatty acid synthesis and suggested the potential of JA as a natural medicinal agent to control lipogenesis in humans. HighlightsO_LIJerusalem artichoke extracts reduce the expression of FASN and ACACA genes at the mRNA and protein levels C_LIO_LITopoisomerase II regulates FASN and ACACA gene expression C_LIO_LIJerusalem artichoke extracts and plasma glucose concentrations regulate cellular topoisomerase II protein expression C_LI

cell biology↗

bioRxiv2023

Innate extracellular Hsp70 inflammatory properties are mediated by the interaction of Siglec-E and LOX-1 receptors

Innate immune responses to cell damage-associated molecular patterns induce a controlled degree of inflammation, ideally avoiding the promotion of intense unwanted inflammatory adverse events. When released by damaged cells, Hsp70 can stimulate different responses that range from immune activation to immune suppression. The effects of Hsp70 are mediated through innate receptors expressed primarily by myeloid cells, such as dendritic cells (DCs). The regulatory innate receptors that bind to extracellular mouse Hsp70 (mHsp70) are not fully characterized, and neither are their potential interactions with activating innate receptors. Here, we describe that extracellular mHsp70 interacts with a receptor complex formed by inhibitory Siglec-E and activating LOX-1 on DCs. We also find that this interaction takes place within lipid microdomains, and Siglec-E acts as a negative regulator of LOX-1-mediated innate activation upon mHsp70 or oxidized LDL binding. Thus, HSP70 can both bind to and modulate the interaction of inhibitory and activating innate receptors on the cell surface. These findings add another dimension of regulatory mechanism to how self-molecules contribute to dampening of exacerbated inflammatory responses.

immunology↗

bioRxiv2021

Extracellular Hsp90α Detoxifies β-Amyloid Fibrils Through an NRF2 and Autophagy Dependent Pathway

We have investigated the role of extracellular Heat shock protein 90 alpha (eHsp90) in conferring protection of neuronal cells against fibrillary amyloid beta (f-A{beta}1-42) toxicity mediated by microglial cells. Formation of f-A{beta}1-42 plaques leads to neurotoxic inflammation, a critical pathological feature of Alzheimers Disease. We observed increased uptake and clearance of internalized f-A{beta}1-42 by microglial cells treated with eHsp90, an effect associated with activation of NRF2 (NF-E2-related factor 2) - mediated autophagy. eHsp90 thus mitigated the neuronal toxicity of f-A{beta}1-42-activated microglia. In addition, eHsp90 facilitated f-A{beta}1-42 engulfment by microglial cells in vitro. In summary, eHsp90 triggers NRF2-mediated autophagy in microglia and thus protects against the neurotoxic effects of f-A{beta}1-42.

cell biology↗

bioRxiv2021

Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans [1, 2]. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models [3, 4]. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

genetics↗

bioRxiv2020

BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase II{beta} (TOP2B)-mediated DNA strand break and the activation of DNA damage response signaling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1 associated ring domain 1 (BARD1) complex, in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this event is essential for productive transcription. Our in vitro biochemical analyses showed TOP2B and BARD1 interaction and colocalization in the EGR1 transcription start site (TSS) and that the BRCA1-BARD1 complex ubiquitinates TOP2B, which appears to stabilize TOP2B protein in the cell and binding to DNA. Intriguingly, BRCA1 phosphorylation at S1524 controls this interaction. In addition, genomic analyses indicated colocalization between TOP2B and BRCA1 in a large number of protein-coding genes. Together, these findings reveal the novel function of the BRCA1-BARD1 complex in gene expression and in the regulation of TOP2B during Pol II transcription. Significance StatementMaintaining genomic integrity against cellular and extracellular genotoxic elements is essential for normal cell growth and function. Recent studies indicated that stimulus-induced transcription provokes topoisomerase II{beta}-mediated DNA strand break and DNA damage response signaling, requiring DNA repair to be coupled with transcription. Here, we present a novel role for the BRCA1-BARD1 complex in regulating the transcription of serum-inducible genes and the stability of topoisomerase II{beta}. The mechanism involving topoisomerase II{beta} ubiquitination by the BRCA1-BARD1 complex and the phosphorylation of BRCA1 S1524 upon transcriptional activation appears to function as a switch to the reaction. Our findings provide the first evidence of functional interaction between the BRCA1-BARD1 complex and topoisomerase II{beta} in transcription in humans.

molecular biology↗

bioRxiv2019

Chemogenomic screening Identifies the Hsp70 Co-chaperone HDJ2 as a Hub for Anticancer Drug Resistance

Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 is regulated by a suite of co-chaperone molecules that bring \"clients\" to the primary chaperone for efficient folding. Therefore, rather than targeting Hsp70 itself, here we have examined the feasibility of inhibiting the co-chaperone HDJ2, a member of the J domain protein family, as a novel anticancer strategy. We found HDJ2 to be upregulated in a variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the NIH Approved Oncology collection for chemical-genetic interactions with loss of HDJ2 in cancer. 41 compounds showed strong synergy with HDJ2 loss, whereas 18 dramatically lost potency. Several of these hits were validated using a HDJ2 inhibitor (116-9e) in castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, these results confirmed that HDJ2 is a hub for anticancer drug resistance and that HDJ2 inhibition may be a potent strategy to sensitize cancer cells to current and future therapeutics.\n\nGraphical AbstractHDJ2 knockout or inhibition via small molecule impacts cellular resistance to anticancer therapeutics.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/818427v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (86K):\norg.highwire.dtl.DTLVardef@cc1f42org.highwire.dtl.DTLVardef@1184229org.highwire.dtl.DTLVardef@19f9b8aorg.highwire.dtl.DTLVardef@19c9037_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

SCAND1 suppresses CDC37 gene transcription by repressing MZF1

CDC37 increases the stability of HSP90 client proteins and is essential for numerous intracellular oncogenic signaling pathways. Elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SCAN transcription factor family- MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for MZF1 were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcrption and reduced tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc-fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in CDC37 gene, negatively regulated CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1.

molecular biology↗

bioRxiv2017

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation

Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.

biochemistry↗