Biology subjects

Wu, L. J.

Publications and source records attributed to Wu, L. J..

9 recordsLinked to original sources

bioRxiv2025

EXPRESSION OF CONJUGATION GENES IS CONTROLLED BY PROCESSIVE ANTITERMINATION AND A NOVEL ZIPPER-TYPE TRANSCRIPTIONAL ATTENUATION MECHANISM

Proper expression of genes clustered in operons, particularly large operons, can be complex, often consisting of multiple regulatory switches. The conjugation operon present on the Bacillus subtilis conjugative plasmid pLS20 is over 32 kb long. This operon starts with a 456 nt leader region, which is followed by the first two genes of the operon, encoding a two-component processive antitermination system. Here, we demonstrate that the long leader region encodes a transcriptional attenuator that we named cATTpLS20. In vivo and in vitro analyses showed that the attenuator is composed of three segments: a stem-loop structure with a long imperfect stem that is preceded by a sequence that may form a weak stem-loop and followed by a sequence that may form an intrinsic terminator. Sequences of the upstream stem loop and downstream terminator are complementary, allowing further extension of the long stem, thereby generating an antiterminator conformation. Based on similarity with a zipper we coined this the zipper-type attenuator. Similar zipper-type attenuators are present upstream of the first genes of conjugation operons of all pLS20 family plasmids, as well as on many other conjugative plasmids in Gram-positive bacteria, suggesting that a common attenuation mechanism regulates expression of many conjugative operons. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/689506v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a86a7eorg.highwire.dtl.DTLVardef@e787f8org.highwire.dtl.DTLVardef@8fdb75org.highwire.dtl.DTLVardef@90f842_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Combination of Imipridone ONC201 or ONC206 with Temozolomide and Radiotherapy in triple ITR therapy reduces intracranial tumor burden and prolongs survival in an orthotopic wild-type IDH GBM mouse model

Glioblastoma remains the most lethal common primary brain tumor in adults with limited therapeutic options. TIC10/ONC201, a first-in-class imipridone we discovered, achieved meaningful therapeutic effects in phase I/II trials in patients with diffuse gliomas harboring H3K27M mutations, and currently the drug is in randomized phase III testing (ACTION trial; NCT05580562). ONC201 targets mitochondrial protease ClpP to disrupt oxidative phosphorylation and trigger the integrated stress response (ISR), TRAIL/DR5, and tumor cell death. We hypothesized that ONC201 and its analogue ONC206 synergize with temozolomide (TMZ) and ionizing radiation (IR), standard-of-care glioblastoma therapies. ONC201 enhances TMZ or IR-induced apoptosis, and cytotoxicity. ClpP-silencing suppresses ONC201-induced cytotoxicity but not TMZ or RT. Both ONC201 and ONC206 reduce expression of TMZ-resistance mediator MGMT. Suppression of MGMT protein was observed in H3K27M-mutated DIPG cell lines following treatment with ONC201 or ONC206 with or without TMZ. Cytokine profiling indicates distinct ONC201 alterations relative to TMZ suggesting distinct anti-tumor immune mechanisms. Triple IR+TMZ+ONC201 (ITR) therapy prolongs median survival to 123 days with a tail on survival curve (3-of-7 mice alive beyond 200-days) in an orthotopic U251 GBM model versus ONC201 (44-days; p=0.000197), IR (63-days; p=0.0012), TMZ (78-days; p=0.0354), ONC201+IR (55-days; p=0.0004), ONC201+TMZ (80-days; p=0.0041) and IR+TMZ (103-days; p>0.05). By 231-days, the only surviving mice were in IRT group. Our results support investigation of ONC201/ONC206 in combination with TMZ and IR (ITR) in GBM or H3K27M mutated diffuse glioma therapy.

cancer biology↗

bioRxiv2024

TRAIL receptor agonist TLY012 in combination with PD-1 inhibition promotes tumor regression in an immune-competent mouse model of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) has an immunosuppressed, apoptosis-resistant phenotype. TLY012 is a pegylated recombinant Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL), an orphan drug for chronic pancreatitis and systemic sclerosis. Innate immune TRAIL signaling suppresses cancer. We hypothesized that combination of immune checkpoint-blocking anti-PD-1 antibody and TLY012 would have synergistic anti-tumor efficacy in immune-competent PDAC-bearing mice. PDAC tumor-bearing C57Bl/6 mice treated 10 mg/kg anti-mouse PD-1 antibody twice weekly and 10 mg/kg TLY012 three times weekly had reduced tumor growth and tumor volume at 70 days compared to either drug alone (all p<0.005). B-cell activating factor (BAFF), which promotes PDAC tumors, decreased to 44% of control mice with dual treatment at 7 days and remained decreased at 3 months. Long-term dual treatment showed the highest levels of proinflammatory cytokines interferon gamma (average 5.6 times control level, p=0.046), CCL5 (average 14.1 times control level, p=0.048), and interleukin-3 (IL-3, average 71.1 times control level, p=0.0053). Flow cytometry showed trends toward decreased circulating regulatory T cells, increased NK cells, and a higher proportion of CD8+ T cells within tumors in dual treatment group. In summary, combination of anti-PD-1 and TLY012 prevented growth of PDAC in an immunocompetent mouse model while increasing tumor-infiltrating CD8+ T cells, decreasing circulating T-regulatory cells and altering cytokine expression of CCL5, interferon gamma and IL-3 to promote proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates changes in immune cell and cytokine levels to induce a more proinflammatory immune environment that contributes to decreased PDAC tumor growth.

cancer biology↗

bioRxiv2024

Synergistic combination therapy with ONC201 or ONC206, Enzalutamide and Darolutamide in preclinical studies of castration-resistant prostate cancer

Androgen receptor (AR) signaling plays a primary role in prostate cancer progression. Non-steroidal anti- androgens (NSAA) including enzalutamide, and apalutamide have been used to treat patients with advanced disease. However, patients with metastatic castration-resistant prostate cancer (mCPRC) develop resistance, resulting in limited overall survival benefit. Darolutamide is a novel next-generation androgen receptor- signaling inhibitor that is FDA approved for non-metastatic castration resistant prostate cancer (nmCRPC). Imipridone ONC201/TIC10 is first-in-class small molecule that activates the integrated stress response (ISR) and upregulates TNF-related apoptosis-inducing ligand (TRAIL). Our study investigates ISR and AR signaling in anti-tumor efficacy with ONC201 and enzalutamide or darolutamide against mCRPC cells. mCRPC cell lines 22RV1, LNCaP, DU145 and PC3 were treated with ONC201, darolutamide, and enzalutamide as single agents or in combinations. Combinations of ONC201 and darolutamide or enzalutamide demonstrated synergistic effects in mCRPC cells. Combinations of ONC201 and darolutamide or enzalutamide reduced PSA levels in LNCaP cells and induced of ATF4 in both LNCaP and 22RV1 cell lines. Darolutamide synergized with ONC201 regardless of AR status or castration sensitivity in vitro. Flow cytometric analysis showed increased intra-tumoral NK cells in mice treated with ONC201 and combination of ONC201 and darolutamide. Trends of increased TRAIL activation within NK cells were also observed in treatment groups. ONC201 and darolutamide demonstrated anti-tumor effects in vivo in the 22RV1 CRPC model. Our results prompt further translational and clinical studies with imipridones ONC201 or ONC201 in combination with enzalutamide or darolutamide for treatment of castrate resistant advanced or metastatic prostate cancer.

cancer biology↗

bioRxiv2024

Heterochromatin spreading in cancer cells through HDAC7 mediated histone H3.3 landscape reprogramming.

Class IIa histone deacetylases (HDACs) are a family of enzymes with minimal histone deacetylase activity but can function as multi-protein interaction hubs. Here we demonstrate the expression of HDAC7, a Class IIa HDAC family member, in glioblastoma tumor tissue from 84 patients, patient-derived glioma stem cells (GSCs) from six patients, and pediatric diffuse pontine glioma (DIPG) cells from three patients. HDAC7 binds to Histone H3.3 and interacts with H3.3 and HIRA on chromatin. Targeted downregulation of HDAC7 expression with a subtype-specific siRNA inhibits the interaction of H3.3 with HIRA while increasing the association of H3.3 with DAXX and H3K9me3. This results in H3.3 being deposited on H3K9me3+/DAPI+ heterochromatin nuclear foci. Inhibition of HDAC7 triggers H3K9me3+ heterochromatin spreading, increased H3K9me3 binding in the cancer genome, and significant alterations in gene expression. Using single molecule DNA fiber approach, we show that HDAC7 inhibition results in a significant increase in replication fork speed without affecting fork symmetry. This altered replication fork speed leads to replication stress, evidenced by phosphorylation of RPA2 and impact on global DNA synthesis, resulting in reduced EdU incorporation. Finally, HDAC7 depletion leads to reduced BRCA2 expression and increased sensitivity of cancer cells to DNA damaging agents. Taken together, these studies uncover the involvement of HDAC7 in the euchromatic H3.3 chaperone network and the effect of HDAC7 depletion on chromatin dynamics, inducing epigenetic restriction and DNA damage in cancer cells.

cancer biology↗

bioRxiv2022

Mircubactin B rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis.

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a complex protein machinery called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg2+ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening >2,000 extracts from a diverse collection of actinobacteria, we identified a new compound, mirubactin B, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed by synthesising mirubactin B. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin B rescuing activity is not due simply to iron sequestration. The results demonstrate a novel antibacterial compound and add to growing evidence that bacterial siderophores have a range of activities beyond simple iron sequestration.

microbiology↗

bioRxiv2021

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.

microbiology↗

bioRxiv2021

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc

ATP and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16-bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (i) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (ii) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

microbiology↗

bioRxiv2019

Geometric principles underlying the proliferation of a model cell system

Wall deficient variants of many bacteria, called L-forms, divide by a simple mechanism that does not depend on the complex FtsZ-based cell division machine. We have used microfluidic systems to probe the growth, chromosome cycle and division mechanism of Bacillus subtilis L-forms. The results show that forcing cells into a narrow linear configuration greatly improves the efficiency of cell growth and chromosome segregation. This reinforces the view that L-form division is driven by an excess accumulation of surface area over volume. Cell geometry was also found to play a dominant role in controlling the relative positions and movement of segregating chromosomes. The presence of the nucleoid appears to influence division both via a cell volume effect and by nucleoid occlusion, even in the absence of the FtsZ machine. Overall, our results emphasise the importance of geometric effects for a range of critical cell functions and are of relevance for efforts to develop artificial or minimal cell systems.

microbiology↗