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Immunity Apr 2024Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize...
Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5' exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2',3'-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.
PubMed: 38697119
DOI: 10.1016/j.immuni.2024.04.010 -
Scientific Reports Apr 2024Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial...
Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.
Topics: Humans; Protein Kinases; HeLa Cells; Ubiquitin-Protein Ligases; Phosphorylation; Ubiquitin; Adenosine Triphosphate; Cyclohexylamines; Pyrimidines
PubMed: 38565869
DOI: 10.1038/s41598-024-58285-3 -
Current Issues in Molecular Biology Feb 2024Ultraviolet (UV) radiation plays a crucial role in the development of melanoma and non-melanoma skin cancers. The types of UV radiation are differentiated by wavelength:... (Review)
Review
Ultraviolet (UV) radiation plays a crucial role in the development of melanoma and non-melanoma skin cancers. The types of UV radiation are differentiated by wavelength: UVA (315 to 400 nm), UVB (280 to 320 nm), and UVC (100 to 280 nm). UV radiation can cause direct DNA damage in the forms of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). In addition, UV radiation can also cause DNA damage indirectly through photosensitization reactions caused by reactive oxygen species (ROS), which manifest as 8-hydroxy-2'-deoxyguanine (8-OHdG). Both direct and indirect DNA damage can lead to mutations in genes that promote the development of skin cancers. The development of melanoma is largely influenced by the signaling of the melanocortin one receptor (MC1R), which plays an essential role in the synthesis of melanin in the skin. UV-induced mutations in the BRAF and NRAS genes are also significant risk factors in melanoma development. UV radiation plays a significant role in basal cell carcinoma (BCC) development by causing mutations in the Hedgehog (Hh) pathway, which dysregulates cell proliferation and survival. UV radiation can also induce the development of squamous cell carcinoma via mutations in the TP53 gene and upregulation of MMPs in the stroma layer of the skin.
PubMed: 38534742
DOI: 10.3390/cimb46030126 -
Heliyon Mar 2024The sensitivity of crops to ultraviolet B (UVB, 280-315 nm) radiation varies significantly. Plants' sensitivity to UVB is heavily influenced by the activity of the... (Review)
Review
The sensitivity of crops to ultraviolet B (UVB, 280-315 nm) radiation varies significantly. Plants' sensitivity to UVB is heavily influenced by the activity of the enzyme cyclobutane pyrimidine dimer (CPD) photolyase, which fixes UVB-induced CPDs. Crops grown in tropical areas with high level of UVB radiation, like from Africa and ssp. rice from Bengal, are more sensitive to UVB radiation and could suffer more as a result of rising UVB levels on the earth's surface. Therefore, creating crops that can withstand high UVB is crucial in tropical regions. There is, however, little information on current techniques for breeding UVB-resistant plants. The most recent techniques for producing UVB-resistant crops are presented in this review. The use of DNA methylation, boosting the antioxidant system, regulating the expression of micro-RNA396, and overexpressing CPD photolyase in transgenic plants are some of the methods that are discussed. CPD photolyase overexpression in transgenic plants is the most popular technique for producing UVB-resistant rice. The study also offers several strategies for creating UVB-resistant plants using gene editing techniques. To feed the world's rapidly expanding population, researchers can use the information from this study to improve food production.
PubMed: 38509919
DOI: 10.1016/j.heliyon.2024.e27806 -
The Journal of Physical Chemistry... Mar 2024The functionalities of proteins rely on protein conformational changes during many processes. Identification of the protein conformations and capturing transitions among...
The functionalities of proteins rely on protein conformational changes during many processes. Identification of the protein conformations and capturing transitions among different conformations are important but extremely challenging in both experiments and simulations. In this work, we develop a machine learning based approach to identify a reaction coordinate that accelerates the exploration of protein conformational changes in molecular simulations. We implement our approach to study the conformational changes of human NTHL1 during DNA repair. Our results identified three distinct conformations: open (stable), closed (unstable), and bundle (stable). The existence of the bundle conformation can rationalize recent experimental observations. Comparison with an NTHL1 mutant demonstrates that a closely packed cluster of positively charged residues in the linker could be a factor to search when screening for genetic abnormalities. Results will lead to a better modulation of the DNA repair pathway to protect against carcinogenesis.
Topics: Humans; Proteins; Protein Conformation; Molecular Dynamics Simulation; Deoxyribonuclease (Pyrimidine Dimer)
PubMed: 38483510
DOI: 10.1021/acs.jpclett.4c00161 -
Plant Physiology Apr 2024Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants...
Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.
Topics: Chloroplasts; Deoxyribodipyrimidine Photo-Lyase; Ultraviolet Rays; Oryza; Plant Proteins; Pyrimidine Dimers; Poaceae; Amino Acid Sequence; Protein Transport
PubMed: 38345835
DOI: 10.1093/plphys/kiae060 -
Chemical Science Feb 2024Charge separation is one of the most common consequences of the absorption of UV light by DNA. Recently, it has been shown that this process can enable efficient...
Charge separation is one of the most common consequences of the absorption of UV light by DNA. Recently, it has been shown that this process can enable efficient self-repair of cyclobutane pyrimidine dimers (CPDs) in specific short DNA oligomers such as the GAT[double bond, length as m-dash]T sequence. The mechanism was characterized as sequential electron transfer through the nucleobase stack which is controlled by the redox potentials of nucleobases and their sequence. Here, we demonstrate that the inverse sequence T[double bond, length as m-dash]TAG promotes self-repair with higher quantum yields (0.58 ± 0.23%) than GAT[double bond, length as m-dash]T (0.44 ± 0.18%) in a comparative study involving UV-irradiation experiments. After extended exposure to UV irradiation, a photostationary equilibrium between self-repair and damage formation is reached at 33 ± 13% for GAT[double bond, length as m-dash]T and at 40 ± 16% for T[double bond, length as m-dash]TAG, which corresponds to the maximum total yield of self-repair. Molecular dynamics and quantum mechanics/molecular mechanics (QM/MM) simulations allowed us to assign this disparity to better stacking overlap between the G and A bases, which lowers the energies of the key A˙G˙ charge transfer state in the dominant conformers of the T[double bond, length as m-dash]TAG tetramer. These conformational differences also hinder alternative photorelaxation pathways of the T[double bond, length as m-dash]TAG tetranucleotide, which otherwise compete with the sequential electron transfer mechanism responsible for CPD self-repair. Overall, we demonstrate that photoinduced electron transfer is strongly dependent on conformation and the availability of alternative photodeactivation mechanisms. This knowledge can be used in the identification and prediction of canonical and modified DNA sequences exhibiting efficient electron transfer. It also further contributes to our understanding of DNA self-repair and its potential role in the photochemical selection of the most photostable sequences on the early Earth.
PubMed: 38332835
DOI: 10.1039/d3sc04971j -
Scientific Reports Feb 2024Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant...
Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).
Topics: DNA Repair; Arabidopsis; Pyrimidinones; Excision Repair; Pyrimidine Dimers; DNA Damage; Receptors, Antigen, T-Cell; Ultraviolet Rays
PubMed: 38332020
DOI: 10.1038/s41598-024-53472-8 -
Scientific Reports Jan 2024DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη...
DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from 'variant of uncertain significance' to 'likely pathogenic mutant', bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.
Topics: Humans; Middle Aged; DNA Damage; Mutation, Missense; Proline; Pyrimidine Dimers; Ultraviolet Rays; Xeroderma Pigmentosum; Female
PubMed: 38212351
DOI: 10.1038/s41598-023-51120-1 -
International Journal of Molecular... Dec 2023Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence...
Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence derivatives (DVs). While CPDs usually occur at higher frequency than DVs, recent studies have shown that the latter display superior selectivity and significant stability in interaction with the human DNA/topoisomerase 1 complex (TOP1). With the aim to deeply investigate the mechanism of interaction of DVs with TOP1, we report here four all-atom molecular dynamic simulations spanning one microsecond. These simulations are focused on the stability and conformational changes of two DNA/TOP1-DV complexes in solution, the data being compared with the biomimetic thymine dimer counterparts. Results from root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) analyses unequivocally confirmed increased stability of the DNA/TOP1-DV complexes throughout the simulation duration. Detailed interaction analyses, uncovering the presence of salt bridges, hydrogen bonds, water-mediated interactions, and hydrophobic interactions, as well as pinpointing the non-covalent interactions within the complexes, enabled the identification of specific TOP1 residues involved in the interactions over time and suggested a potential TOP1 inhibition mechanism in action.
Topics: Humans; Molecular Dynamics Simulation; DNA Topoisomerases, Type I; Biomimetics; DNA Adducts; Data Interpretation, Statistical; Pyrimidine Dimers
PubMed: 38203410
DOI: 10.3390/ijms25010234