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1.
Front Cell Dev Biol ; 9: 760226, 2021.
Article in English | MEDLINE | ID: mdl-34950659

ABSTRACT

The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells.

2.
Chemistry ; 25(8): 2019-2024, 2019 Feb 06.
Article in English | MEDLINE | ID: mdl-30427558

ABSTRACT

Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template-based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia-inducible transcription factor prolyl hydroxylase, PHD2.


Subject(s)
Prolyl Hydroxylases , Prolyl-Hydroxylase Inhibitors , Ribosomes/drug effects , Carrier Proteins/antagonists & inhibitors , Drug Design , Humans , Nuclear Proteins/antagonists & inhibitors , Prolyl Hydroxylases/metabolism , Prolyl-Hydroxylase Inhibitors/chemistry , Prolyl-Hydroxylase Inhibitors/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology , Ribosomes/metabolism , Structure-Activity Relationship , Substrate Specificity
3.
Sci Rep ; 7(1): 700, 2017 04 06.
Article in English | MEDLINE | ID: mdl-28386087

ABSTRACT

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.


Subject(s)
Epithelial Cells/metabolism , Epithelial Sodium Channels/genetics , Gene Transfer Techniques , Nanoparticles , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Transduction, Genetic , Cell Line , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis/therapy , Gene Knockdown Techniques , Gene Silencing , Genetic Therapy , Liposomes/chemistry , Microscopy, Confocal , Peptides/chemistry , RNA Interference , RNA, Small Interfering/chemistry , Transfection
4.
Am J Hum Genet ; 100(3): 506-522, 2017 Mar 02.
Article in English | MEDLINE | ID: mdl-28257692

ABSTRACT

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.


Subject(s)
Ribosomal Proteins/genetics , Ribosomes/genetics , Autism Spectrum Disorder/genetics , Carrier Proteins/genetics , Cells, Cultured , Child , Child, Preschool , Codon/genetics , Developmental Disabilities/genetics , Exome , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Genetic Variation , Hearing Loss/genetics , Humans , Intellectual Disability/genetics , Male , Microcephaly/genetics , Mutation , Mutation, Missense , Nuclear Proteins/genetics , Oxidative Stress , Protein Biosynthesis/genetics , Sequence Alignment , Sequence Analysis, DNA
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