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The Journal of Biological Chemistry Jun 2024The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the...
The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration (AMD) and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.
PubMed: 38876307
DOI: 10.1016/j.jbc.2024.107467 -
The Journal of Biological Chemistry Jun 2024Theta-mediated end-joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta...
Theta-mediated end-joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend single-stranded DNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both single-stranded DNA and RNA substrates by unimolecular stem loop synthesis initiated by only two 3' terminal base-pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem loop synthesis competes with bimolecular end-joining, even when a longer terminal microhomology for end-joining is available. Both reactions are partially suppressed by the ssDNA binding protein RPA. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare amongst human DNA polymerases but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.
PubMed: 38876299
DOI: 10.1016/j.jbc.2024.107461 -
Neoplasia (New York, N.Y.) Jun 2024Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by...
Increased mutational burden and EBV load have been revealed from normal tissues to Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs). BPLF1, encoded by EBV, is a lytic cycle protein with deubiquitinating activity has been found to participate in disrupting repair of DNA damage. We first confirmed that BPLF1 gene in gastric cancer (GC) significantly increased the DNA double strand breaks (DSBs). Ubiquitination mass spectrometry identified histones as BPLF1 interactors and potential substrates, and co-immunoprecipitation and in vitro experiments verified that BPLF1 regulates H2Bub by targeting Rad6. Over-expressing Rad6 restored H2Bub but partially reduced γ-H2AX, suggesting that other downstream DNA repair processes were affected. mRNA expression of BRCA2 were significantly down-regulated by next-generation sequencing after over-expression of BPLF1, and over-expression of p65 facilitated the repair of DSBs. We demonstrated BPLF1 may lead to the accumulation of DSBs by two pathways, reducing H2B ubiquitination (H2Bub) and blocking homologous recombination which may provide new ideas for the treatment of gastric cancer.
PubMed: 38875930
DOI: 10.1016/j.neo.2024.101012 -
Cancer Control : Journal of the Moffitt... 2024The present study aimed to evaluate the frequencies of , and mutations and their possible associations with clinicopathological features in 249 Moroccan patients with...
OBJECTIVES
The present study aimed to evaluate the frequencies of , and mutations and their possible associations with clinicopathological features in 249 Moroccan patients with colorectal cancer (CRC).
METHODS
A retrospective investigation of a cohort of formalin-fixed paraffin-embedded tissues of 249 patients with CRC was screened for // mutations using Idylla™ technology and pyrosequencing.
RESULTS
, and mutations were revealed in 46.6% (116/249), 5.6% (14/249), and 2.4% (6/249) of patients. exon 2 mutations were identified in 87.9% of patients (102/116). G12D and G12 C were the most frequent, at 32.8% and 12.93%, respectively. Among the patients with exon 2 wild-type (wt), 27.6% (32/116) harbored additional mutations. Concurrent mutations were identified in 9.5% (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12 A and Q61H), and one in codon 13 (G13D and Q61 L). Among the exon 2 wt patients, 64.3% (9/14) harbored additional mutations. Concurrent mutations were identified in 28.6% (4/14) of -mutant patients. Since 3.2% wt were identified with mutations, concomitant and mutations were identified in 2.4% (6/249) of patients. mutations were higher in the >50-year-old age-group ( = .031), and the tumor location was revealed to be significantly associated with mutations ( = .028) predominantly in left colon (27.5%) and colon (42.2%) locations. mutations were most prevalent in the left colon (42.8%) and in well-differentiated tumors (64.2%).
CONCLUSION
Detection of mutations, particularly the G12 C subtype, may be significant for patients with CRC and has possible therapeutic implications. However, rare concomitant mutations in CRC patients suggest that each individual may present distinct therapeutic responses. testing alongside the identification of other affected genes in the same patient will make the treatments even more personalized by contributing more accurately to the clinical decision process. Overall, early diagnosis using novel molecular techniques may improve the management of CRC by providing the most efficient therapies for Moroccan patients.
Topics: Humans; Proto-Oncogene Proteins B-raf; Colorectal Neoplasms; Male; Female; Proto-Oncogene Proteins p21(ras); Membrane Proteins; Middle Aged; GTP Phosphohydrolases; Morocco; Mutation; Retrospective Studies; Aged; Adult; Aged, 80 and over; DNA Mutational Analysis
PubMed: 38875469
DOI: 10.1177/10732748241262179 -
Science Advances Jun 2024
PubMed: 38875346
DOI: 10.1126/sciadv.adq7259 -
PloS One 2024The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on...
The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.
Topics: DNA; Oxidative Stress; Nucleic Acid Denaturation; Temperature; Oxidation-Reduction; DNA Damage; Hydrogen-Ion Concentration; Guanine; Electrochemical Techniques; Adenine
PubMed: 38875261
DOI: 10.1371/journal.pone.0305590 -
PloS One 2024Identifying and describing molecular alterations in tumors has become common with the development of high-throughput sequencing. However, DNA sequencing in rare tumors,... (Review)
Review
BACKGROUND
Identifying and describing molecular alterations in tumors has become common with the development of high-throughput sequencing. However, DNA sequencing in rare tumors, such as ovarian adult granulosa cell tumor (aGCT), often lacks statistical power due to the limited number of cases in each study. Questions regarding personalized treatment or prognostic biomarkers for recurrence or other malignancies therefore still need to be elucidated. This scoping review protocol aims to systematically map the current evidence and identify knowledge gaps regarding DNA alterations, actionable variations and prognostic biomarkers in aGCT.
METHODS
This scoping review will be conducted based on Arksey and O'Malley's methodological framework and later modifications by JBI Evidence Synthesis. The protocol complies with Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews. All original publications describing molecular alterations of aGCT will be included. The search will be performed in May 2024 in the following databases: MEDLINE (Ovid), Embase (Ovid), Web of Science Core Collection and Google Scholar (100-top ranked).
DISCUSSION
This scoping review will identify knowledge and gaps in the current understanding of the molecular landscape of aGCT, clinical trials on actionable variations and priorities for future research. As aGCT are rare, a possible limitation will be the small sample sizes and heterogenic study settings.
SCOPING REVIEW REGISTRATION
The review protocol is registered at Open Science Framework under https://doi.org/10.17605/OSF.IO/PX4MF.
Topics: Humans; Granulosa Cell Tumor; Female; Ovarian Neoplasms; Biomarkers, Tumor
PubMed: 38875223
DOI: 10.1371/journal.pone.0303989 -
STAR Protocols Jun 2024Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA...
Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al..
PubMed: 38875114
DOI: 10.1016/j.xpro.2024.103128 -
Journal of Radiation Research Jun 2024Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell...
Recently, biomolecular condensates formed through liquid-liquid phase separation have been widely reported to regulate key intracellular processes involved in cell biology and pathogenesis. BRD4 is a nuclear protein instrumental to the establishment of phase-separated super-enhancers (SEs) to direct the transcription of important genes. We previously observed that protein droplets of BRD4 became hydrophobic as their size increase, implying an ability of SEs to limit the ionization of water molecules by irradiation. Here, we aim to establish if SEs confer radiation resistance in cancer cells. We established an in vitro DNA damage assay that measures the effect of radicals provoked by the Fenton reaction on DNA integrity. This revealed that DNA damage was markedly reduced when BRD4 underwent phase separation with DNA. Accordingly, co-focal imaging analyses revealed that SE foci and DNA damage foci are mutually exclusive in irradiated cells. Lastly, we observed that the radioresistance of cancer cells was significantly reduced when irradiation was combined with ARV-771, a BRD4 de-stabilizer. Our data revealed the existence of innately radioresistant genomic regions driven by phase separation in cancer cells. The disruption of these phase-separated components enfolding genomic DNA may represent a novel strategy to augment the effects of radiotherapy.
PubMed: 38874522
DOI: 10.1093/jrr/rrae044 -
Aging Jun 2024Prior studies showed increased age acceleration (AgeAccel) is associated with worse cognitive function among old adults. We examine the associations of childhood,...
Prior studies showed increased age acceleration (AgeAccel) is associated with worse cognitive function among old adults. We examine the associations of childhood, adolescence and midlife cognition with AgeAccel based on DNA methylation (DNAm) in midlife. Data are from 359 participants who had cognition measured in childhood and adolescence in the Child Health and Development study, and had cognition, blood based DNAm measured during midlife in the Disparities study. Childhood cognition was measured by Raven's Progressive Matrices and Peabody Picture Vocabulary Test (PPVT). Adolescent cognition was measured only by PPVT. Midlife cognition included Wechsler Test of Adult Reading (WTAR), Verbal Fluency (VF), Digit Symbol (DS). AgeAccel measures including Horvath, Hannum, PhenoAge, GrimAge and DunedinPACE were calculated from DNAm. Linear regressions adjusted for potential confounders were utilized to examine the association between each cognitive measure in relation to each AgeAccel. There are no significant associations between childhood cognition and midlife AgeAccel. A 1-unit increase in adolescent PPVT, which measures crystalized intelligence, is associated with 0.048-year decrease of aging measured by GrimAge and this association is attenuated after adjustment for adult socioeconomic status. Midlife crystalized intelligence measure WTAR is negatively associated with PhenoAge and DunedinPACE, and midlife fluid intelligence measure (DS) is negatively associated with GrimAge, PhenoAge and DunedinPACE. AgeAccel is not associated with VF in midlife. In conclusion, our study showed the potential role of cognitive functions at younger ages in the process of biological aging. We also showed a potential relationship of both crystalized and fluid intelligence with aging acceleration.
PubMed: 38874516
DOI: 10.18632/aging.205943