-
Cells Oct 2023Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation....
Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity.
Topics: Humans; Sumoylation; Consensus; HeLa Cells; Microtubule-Associated Proteins; Microtubules
PubMed: 37947624
DOI: 10.3390/cells12212545 -
Plant Disease Nov 2023Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities,...
Trichosanthis fructus is one of the most common medicinal plants in China. In September 2022, T. fructus fruit showed black necrotic spots and surface irregularities, with water-soaked lesions (Fig 1). The affected T. fructus fruit (five weeks after blossom) were located in a field in Huai'an Municipality, Jiangsu Province (33.85°N, 119.00°E). The incidence was approximately 50%, causing great losses in fruit production. To isolate the causal agent, two symptomatic fruit from different plants were surface-disinfested with 75% (v/v) ethanol for 1 min, washed three times with sterile distilled water, and cultured on Nutrient agar (NA) plates at 28°C for 24 h. The obtained colonies were light yellow and transferred to fresh NA plates using the conventional repetitive streaking technique to obtain pure cultures. The purified bacterial cells were rod shaped, 1.64 to 2.47 μm long (n = 45), and 0.58 to 0.74 μm wide (n = 45) (Figure S2). Three isolates were used for further characterization. Biochemical tests indicated that the three isolates were Gram negative. DNA was extracted from the three bacterial isolates and used to amplify the16S rRNA (27F/1492R primers) and partial gyrB (UP1/Up2r primers) genes (Marchesi et al. 1998; Yamamoto and Harayama 1995). PCR products were purified using the DNA Clean-up Kit (CW2301, CWBIO), ligated into the PMD-19 vector (6013, Takara), and sequenced by Beijing Tsingke Biotech. The obtained 16S rRNA (GenBank accessions: OQ923996-OQ923998) and gyrB sequences (OR140942-OR140944) showed the best match, over 99%and 98% identity with 100% coverage to the K. cowanii type strain JCM 10956 (CP019445.1). To fulfill Koch's postulates, pathogenicity tests were conducted on healthy T. fructus fruit. T. fructus fruit showed no wounds or lesions, and were surface disinfected with 75% alcohol. The three isolates were grown in nutrient broth at 200 rpm in 28 oC for 24 h and re-suspended in sterilized ddH2O to OD600 = 0.6~1.0 (108~109cfu/mL). Five μL of bacterial suspension was inoculated into the healthy fruit surface with a sterile knife. For the control experiment, the same volume of sterilized ddH2O was used. In each treatment, four healthy T. fructus fruit were treated. All samples were incubated at 25°C and 75% humidity in a plant incubator (Bluepard, MGC-350BP-2). After 12 days, bacterial fruit blotch symptoms were observed in all the inoculated fruit. The pathogen was recovered from the infected fruit, and its identity was confirmed by 16S rRNA/gyrB sequencing and morphological analysis. To further investigate the pathogenicity, four-week-old T. fructus plant leaves were inoculated with the above three isolated suspension (OD600=0.6~1.0) using the leaf cutting method (Kauffman et al. 1973). Sterilized ddH2O was used as mock control. After 10 days, bacterial blight symptoms were observed in all inoculated leaves. To our knowledge, this is the first report of K. cowanii causing fruit blotch on T. fructus worldwide. This species has been previously associated with acute cholecystitis in humans (Berinson et al. 2020; Petrzik et al. 2021), but it was recently identified as the causal agent of bacterial wilt on patchouli, bacterial blight on soybean, and stalk rot in foxtail millet (Han et al. 2023; Krawczyk and Borodynko-Filas 2020; Zhang et al. 2022). China is the largest producer of T. fructus. This report reveals that K. cowanii has a greater host range than was known. This report will help to better understand the pathogens that affects T. fructus production in China.
PubMed: 37971893
DOI: 10.1094/PDIS-10-23-2048-PDN -
Frontiers in Cell and Developmental... 2024The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome...
The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless-Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless-Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.
PubMed: 38487270
DOI: 10.3389/fcell.2024.1346534 -
Molecular Oncology Feb 2024The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes...
Subcellular localization of PD-L1 and cell-cycle-dependent expression of nuclear PD-L1 variants: implications for head and neck cancer cell functions and therapeutic efficacy.
The programmed cell death 1 ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis is primarily associated with immunosuppression in cytotoxic T lymphocytes (CTLs). However, mounting evidence is supporting the thesis that PD-L1 not only functions as a ligand but mediates additional cellular functions in tumor cells. Moreover, it has been demonstrated that PD-L1 is not exclusively localized at the cellular membrane. Subcellular fractionation revealed the presence of PD-L1 in various cellular compartments of six well-characterized head and neck cancer (HNC) cell lines, including the nucleus. Via Western blotting, we detected PD-L1 in its well-known glycosylated/deglycosylated state at 40-55 kDa. In addition, we detected previously unknown PD-L1 variants with a molecular weight at approximately 70 and > 150 kDa exclusively in nuclear protein fractions. These in vitro findings were confirmed with primary tumor samples from head and neck squamous cell carcinoma (HNSCC) patients. Furthermore, we demonstrated that nuclear PD-L1 variant expression is cell-cycle-dependent. Immunofluorescence staining of PD-L1 in different cell cycle phases of synchronized HNC cells supported these observations. Mechanisms of nuclear PD-L1 trafficking remain less understood; however, proximity ligation assays showed a cell-cycle-dependent interaction of the cytoskeletal protein vimentin with PD-L1, whereas vimentin could serve as a potential shuttle for nuclear PD-L1 transportation. Mass spectrometry after PD-L1 co-immunoprecipitation, followed by gene ontology analysis, indicated interaction of nuclear PD-L1 with proteins involved in DNA remodeling and messenger RNA (mRNA) splicing. Our results in HNC cells suggest a highly complex regulation of PD-L1 and multiple tumor cell-intrinsic functions, independent of immune regulation. These observations bear significant implications for the therapeutic efficacy of immune checkpoint inhibition.
Topics: Humans; B7-H1 Antigen; Cell Cycle; Head and Neck Neoplasms; Squamous Cell Carcinoma of Head and Neck; Vimentin
PubMed: 38103190
DOI: 10.1002/1878-0261.13567 -
Cancer Genomics & Proteomics 2023Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS)...
BACKGROUND/AIM
Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS) technologies have contributed to multi gene panel analysis in clinical practice.
MATERIALS AND METHODS
A total of 2,163 patients were screened for cancer susceptibility, using a solution-based capture method. A panel of 52 genes was used for targeted NGS. The capture-based approach enables computational analysis of CNVs from NGS data. We studied the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and of the non-commercial tool panelcn.MOPS. Additionally, we tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA).
RESULTS
Pathogenic/likely pathogenic variants (P/LP) were identified in 464 samples (21.5%). CNV accounts for 10.8% (50/464) of pathogenic variants, referring to deletion/duplication of one or more exons of a gene. In patients with breast and ovarian cancer, CNVs accounted for 10.2% and 6.8% of pathogenic variants, respectively. In colorectal cancer patients, CNV accounted for 28.6% of pathogenic/likely pathogenic variants.
CONCLUSION
In silico CNV detection tools provide a viable and cost-effective method to identify CNVs from NGS experiments. CNVs constitute a substantial percentage of P/LP variants, since they represent up to one of every ten P/LP findings identified by NGS multigene analysis; therefore, their evaluation is highly recommended to improve the diagnostic yield of hereditary cancer analysis.
Topics: Female; Humans; DNA Copy Number Variations; Genetic Predisposition to Disease; Ovarian Neoplasms; High-Throughput Nucleotide Sequencing; Exons; Genetic Testing
PubMed: 37643779
DOI: 10.21873/cgp.20396 -
BioRxiv : the Preprint Server For... Nov 2023Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their...
Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.
PubMed: 38045421
DOI: 10.1101/2023.11.21.568171 -
BioRxiv : the Preprint Server For... Jan 2024The influence of the metastasis promoting proteins mutant p53 (mtp53) and MDM2 on (CPR) to promote cancer cell survival is understudied. Interactions between the DNA...
The influence of the metastasis promoting proteins mutant p53 (mtp53) and MDM2 on (CPR) to promote cancer cell survival is understudied. Interactions between the DNA repair choice protein 53BP1 and wild type tumor suppressor protein p53 (wtp53) regulates cell cycle control. Cancer cells often express elevated levels of transcriptionally inactive missense mutant p53 (mtp53) that interacts with MDM2 and MDM4/MDMX (herein called MDMX). The ability of mtp53 to maintain a 53BP1 interaction while in the context of interactions with MDM2 and MDMX has not been described. We asked if MDM2 regulates chromatin-based phosphorylation events in the context of mtp53 by comparing the chromatin of T47D breast cancer cells with and without MDM2 in a phospho-peptide stable isotope labeling in cell culture (SILAC) screen. We found reduced phospho-53BP1 chromatin association, which we confirmed by chromatin fractionation and immunofluorescence in multiple breast cancer cell lines. We used the Proximity Ligation Assay (PLA) in breast cancer cell lines and detected 53BP1 in close proximity to mtp53, MDM2, and the DNA repair protein MDC1. Through disruption of the mtp53-MDM2 interaction, by either Nutlin 3a or a mtp53 R273H C-terminal deletion, we uncovered that mtp53 was required for MDM2-53BP1 interaction foci. Our data suggests that mtp53 works with MDM2 and 53BP1 to promote CPR and cell survival.
PubMed: 38328189
DOI: 10.1101/2024.01.20.576487 -
New Biotechnology Sep 2023Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR...
Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on every hairpin being metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which puts a strong demand on oligonucleotide quality. We show how further purification can greatly increase polymerization potential. It was found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than a non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.
Topics: Oligonucleotides; DNA; Nucleic Acid Hybridization; Hybridization, Genetic
PubMed: 37059331
DOI: 10.1016/j.nbt.2023.04.004 -
STAR Protocols Dec 2023Transcription factors (TFs) play a pivotal role in gene expression, and their DNA binding is the prerequisite to instigating gene transcription. Here, we present a...
Transcription factors (TFs) play a pivotal role in gene expression, and their DNA binding is the prerequisite to instigating gene transcription. Here, we present a protocol that exploits the proximity ligation assay technique to measure the DNA-binding activities of TFs in situ at the single-cell resolution. We describe steps for immunostaining with specific antibodies against double-stranded DNA and the TFs of interest, probe incubation, proximity ligation, and signal amplification. We then detail procedures for imaging and image analysis. For complete details on the use and execution of this protocol, please refer to Dai et al. (2015) and Xu et al. (2023)..
Topics: Transcription Factors; Antibodies; Image Processing, Computer-Assisted; Cells, Cultured
PubMed: 37917578
DOI: 10.1016/j.xpro.2023.102692 -
BioRxiv : the Preprint Server For... Jun 2024Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with...
Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the , and genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3'-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.
PubMed: 38895297
DOI: 10.1101/2024.06.03.597111