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MBio Jun 2024Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an...
UNLABELLED
Merkel cell polyomavirus (MCPyV) is a double-stranded tumor virus that is the main causative agent of Merkel cell carcinoma (MCC). The MCPyV large T antigen (LT), an essential viral DNA replication protein, maintains viral persistence by interacting with host Skp1-Cullin 1-F-box (SCF) E3 ubiquitin ligase complexes, which subsequently induces LT's proteasomal degradation, restricting MCPyV DNA replication. SCF E3 ubiquitin ligases require their substrates to be phosphorylated to bind them, utilizing phosphorylated serine residues as docking sites. The MCPyV LT unique region (MUR) is highly phosphorylated and plays a role in multiple host protein interactions, including SCF E3 ubiquitin ligases. Therefore, this domain highly governs LT stability. Though much work has been conducted to identify host factors that restrict MCPyV LT protein expression, the kinase(s) that cooperates with the SCF E3 ligase remains unknown. Here, we demonstrate that casein kinase 1 alpha (CK1α) negatively regulates MCPyV LT stability and LT-mediated replication by modulating interactions with the SCF β-TrCP. Specifically, we show that numerous CK1 isoforms (α, δ, ε) localize in close proximity to MCPyV LT through proximity ligation assays (PLA) and CK1α overexpression mainly resulted in decreased MCPyV LT protein expression. Inhibition of CK1α using short hairpin RNA (shRNA) and treatment of a CK1α inhibitor or an mTOR inhibitor, TORKinib, resulted in decreased β-TrCP interaction with LT, increased LT expression, and enhanced MCPyV replication. The expression level of the gene transcripts is higher in MCPyV-positive MCC, suggesting a vital role of CK1α in limiting MCPyV replication required for establishing persistent infection.
IMPORTANCE
Merkel cell polyomavirus (MCPyV) large tumor antigen is a polyphosphoprotein and the phosphorylation event is required to modulate various functions of LT, including viral replication. Therefore, cellular kinase pathways are indispensable for governing MCPyV polyomavirus infection and life cycle in coordinating with the immunosuppression environment at disease onset. Understanding the regulation mechanisms of MCPyV replication by viral and cellular factors will guide proper prevention strategies with targeted inhibitors for MCPyV-associated Merkel cell carcinoma (MCC) patients, who currently lack therapies.
PubMed: 38940554
DOI: 10.1128/mbio.01117-24 -
G3 (Bethesda, Md.) Jun 2024Elaeis guineensis and E. oleifera are the two species of oil palm. E. guineensis is the most widely cultivated commercial species, and introgression of desirable traits...
Elaeis guineensis and E. oleifera are the two species of oil palm. E. guineensis is the most widely cultivated commercial species, and introgression of desirable traits from E. oleifera is ongoing. We report an improved E. guineensis genome assembly with substantially increased continuity and completeness, as well as the first chromosome-scale E. oleifera genome assembly. Each assembly was obtained by integration of long-read sequencing, proximity ligation sequencing, optical mapping and genetic mapping. High interspecific genome conservation is observed between the two species. The study provides the most extensive gene annotation to date, including 46,697 E. guineensis and 38,658 E. oleifera gene predictions. Analyses of repetitive element families further resolve the DNA repeat architecture of both genomes. Comparative genomic analyses identified experimentally validated small structural variants between the oil palm species and resolved the mechanism of chromosomal fusions responsible for the evolutionary descending dysploidy from 18 to 16 chromosomes.
PubMed: 38918881
DOI: 10.1093/g3journal/jkae135 -
PloS One 2024Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy and a major cause of blindness. RP is caused by several variants of multiple genes, and genetic...
Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy and a major cause of blindness. RP is caused by several variants of multiple genes, and genetic diagnosis by identifying these variants is important for optimizing treatment and estimating patient prognosis. Next-generation sequencing (NGS), which is currently widely used for diagnosis, is considered useful but is known to have limitations in detecting copy number variations (CNVs). In this study, we re-evaluated CNVs in EYS, the main causative gene of RP, identified via NGS using multiplex ligation-dependent probe amplification (MLPA). CNVs were identified in NGS samples of eight patients. To identify potential CNVs, MLPA was also performed on samples from 42 patients who were undiagnosed by NGS but carried one of the five major pathogenic variants reported in Japanese EYS-RP cases. All suspected CNVs based on NGS data in the eight patients were confirmed via MLPA. CNVs were found in 2 of the 42 NGS-undiagnosed RP cases. Furthermore, results showed that 121 of the 661 patients with RP had EYS as the causative gene, and 8.3% (10/121 patients with EYS-RP) had CNVs. Although NGS using the CNV calling criteria utilized in this study failed to identify CNVs in two cases, no false-positive results were detected. Collectively, these findings suggest that NGS is useful for CNV detection during clinical diagnosis of RP.
Topics: Humans; Retinitis Pigmentosa; DNA Copy Number Variations; High-Throughput Nucleotide Sequencing; Female; Male; Eye Proteins; Middle Aged; Adult; Multiplex Polymerase Chain Reaction
PubMed: 38913662
DOI: 10.1371/journal.pone.0305812 -
Analytical Chemistry Jun 2024DNA walker, a type of dynamic DNA device that is capable of moving progressively along prescribed walking tracks, has emerged as an ideal and powerful tool for...
DNA walker, a type of dynamic DNA device that is capable of moving progressively along prescribed walking tracks, has emerged as an ideal and powerful tool for biosensing and bioimaging. However, most of the reported three-dimensional (3D) DNA walker were merely designed for the detection of a single target, and they were not capable of achieving universal applicability. Herein, we reported for the first time the development of a proximity-induced 3D bipedal DNA walker for imaging of low abundance biomolecules. As a proof of concept, miRNA-34a, a biomarker of breast cancer, is chosen as the model system to demonstrate this approach. In our design, the 3D bipedal DNA walker can be generated only by the specific recognition of two proximity probes for miRNA-34a. Meanwhile, it stochastically and autonomously traveled on 3D tracks (gold nanoparticles) via catalytic hairpin assembly (CHA), resulting in the amplified fluorescence signal. In comparison with some conventional DNA walkers that were utilized for living cell imaging, the 3D DNA walkers induced by proximity ligation assay can greatly improve and ensure the high selectivity of bioanalysis. By taking advantage of these unique features, the proximity-induced 3D bipedal DNA walker successfully realizes accurate and effective monitoring of target miRNA-34a expression levels in living cells, affording a universal, valuable, and promising platform for low-abundance cancer biomarker detection and accurate identification of cancer.
PubMed: 38913536
DOI: 10.1021/acs.analchem.4c01483 -
STAR Protocols Jun 2024Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions...
Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts. For complete details on the use and execution of this protocol, please refer to Zhao et al..
PubMed: 38907997
DOI: 10.1016/j.xpro.2024.103149 -
Methods in Molecular Biology (Clifton,... 2024RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases....
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
Topics: In Situ Hybridization; RNA; Humans; Chromogenic Compounds; Colorimetry; Single Molecule Imaging
PubMed: 38907917
DOI: 10.1007/978-1-0716-3918-4_11 -
Analytical Chemistry Jun 2024Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate...
Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS.
Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 10-10 particles/mL, along with a detection limit of 1.1 × 10 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.
PubMed: 38904228
DOI: 10.1021/acs.analchem.4c02066 -
Nature Structural & Molecular Biology Jun 2024In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair...
In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.
PubMed: 38898102
DOI: 10.1038/s41594-024-01339-x -
Journal of Clinical Immunology Jun 2024A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid...
A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.
Topics: Female; Humans; Male; DNA Ligase ATP; Fibroblasts; Homozygote; Molecular Dynamics Simulation; Mutation; Severe Combined Immunodeficiency; Infant
PubMed: 38896336
DOI: 10.1007/s10875-024-01754-1 -
BioRxiv : the Preprint Server For... Jun 2024Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In...
Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes . In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.
PubMed: 38895383
DOI: 10.1101/2024.06.03.597171