Repair Proteins Research Articles

The impact of preoperative treatment on mismatch repair protein and HER2 expression in colorectal cancer: an analysis of paired samples.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, highlighting the necessity for multifaceted treatment strategies, including preoperative treatment (PT), which can enhance surgical outcomes and provide prognostic insights. This study aims to clarify the impact of PT-induced changes in mismatch repair (MMR) and human epidermal growth factor receptor 2 (HER2) expression, potentially informing tailored treatment strategies and improving clinical outcomes for CRC patients. This retrospective study analyzed 120 paired samples from CRC patients who underwent preoperative treatment, comparing pre- and post-treatment specimens. A control group of 60 untreated surgical specimens was also included. Immunohistochemistry assessed MMR proteins (MSH6, MSH2, MLH1, PMS2) and HER2 expression. MSI status was determined in samples with low MMR expression. Compared to pre-treatment samples, post-treatment samples exhibited lower levels of MSH6, MSH2, and total MMR expression, along with higher levels of HER2 expression. However, when compared to the untreated control group, there were no significant differences in the expression of MSH6, total MMR, and HER2. All samples that exhibited weak MMR expression and those that shifted to deficient mismatch repair (dMMR) status following treatment had stable microsatellite status. No clear clinicopathological characteristics or prognostic factors were found to be associated with changes in MMR and HER2 expression, except for the use of fluorouracil or capecitabine, which was related to changes in total MMR scores. ypTNM stage and TRG scores were identified as independent factors affecting disease progression in our study. PT is associated with a reduction in MMR expression, notably for the MSH2 protein, while it does not appear to influence HER2 expression.

A Site-Specific Click Chemistry Approach to Di-Ubiquitylate H1 Variants Reveals Position-Dependent Stimulation of the DNA Repair Protein RNF168.

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2 variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2 showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2 into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2 variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.

Action-At-A-Distance in DNA Mismatch Repair: Mechanistic Insights and Models for How DNA and Repair Proteins Facilitate Long-Range Communication

Many DNA metabolic pathways, including DNA repair, require the transmission of signals across long stretches of DNA or between DNA molecules. Solutions to this signaling challenge involve various mechanisms: protein factors can travel between these sites, loop DNA between sites, or form oligomers that bridge the spatial gaps. This review provides an overview of how these paradigms have been used to explain DNA mismatch repair, which involves several steps that require action-at-a-distance. Here, we describe these models in detail and how current data fit into these descriptions. We also outline regulation steps that remain unanswered in how the action is communicated across long distances along a DNA contour in DNA mismatch repair.

STK19 facilitates the clearance of lesion-stalled RNAPII during transcription-coupled DNA repair.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

CNSC-40. GTP SIGNALING PATHWAY PROMOTES NHEJ REPAIR BY INDUCING NUCLEAR ACTIN POLYMERIZATION IN GLIOBLASTOMA

Abstract OBJECTIVES We recently identified that GTP promotes non-homologous end joining (NHEJ) by triggering a GTP-Rac1-PP5-Abi-1/S323 cascade and we sought to define the molecular mechanisms. METHODS Crispr/Cas9, CDNA overexpression and immunoblot were used to modulate and confirm protein levels. Cellular fractionation and immunofluorescence (IF) were used to determine the localization of proteins. Laser micro-irradiation was used to induce DNA damage and monitor the recruitment of DNA repair proteins. RESULTS We found that GTP-activated protein Rac1 and Abi-1, the two key components of the newly identified pathway, shuttled into the nucleus post-RT in glioblastoma (GBM) cells. These effects were further enhanced by the GTP precursor guanosine but blocked by GTP depletion. Furthermore, Abi-1 and Rac1 accumulated in the nucleus of the cells expressing constitutively active Rac1 but not dominant negative Rac1 after RT, which suggests that Rac1/Abi-1 nuclear translocation is Rac1 activity dependent. We previously discovered that GTP-Rac1 promotes DNA repair by facilitating the dephosphorylation of Abi-1 (S323). Here, we discovered that dephosphomimetic Abi-1 (S323A) but not phosphomimetic Abi-1 (S323D) shuttled into nucleus after RT. Because Rac1 canonically binds to Abi-1 and promotes actin polymerization in the cytosol, we next wanted to determine if RT-induced nuclear Rac1/Abi-1/S323A could enhance nuclear actin polymerization, which could enhance NHEJ repair. We found that Abi-1/S323A but not Abi-1/S323D increases nuclear filament actin formation post-RT and activating the GTP-Rac1-Abi-1/S323A axis (through guanosine supplementation, expression of constitutively active Rac1 or expression of dephosphomimetic Abi-1/S323A) enhanced the recruitment and stability of KU80 at sites of DNA damage, which could be blocked by actin polymerization inhibitor cytochalasin D. Furthermore, inhibiting actin polymerization overcame radiation resistance in GBM cells with an active GTP-Rac1-Abi-1/S323A axis. CONCLUSIONS GTP-Rac1-PP5-Abi-1/S323 cascade promotes NHEJ repair by inducing the nuclear assembly of filament actin and disrupting this regulation could improve treatment responses in GBMs.

TMET-28. RECIPROCAL LINKS BETWEEN METHIONINE METABOLISM, DNA REPAIR AND THERAPY RESISTANCE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is uniformly lethal due to profound treatment resistance. Altered cellular metabolism is a key mediator of GBM treatment resistance. Uptake of the essential sulfur-containing amino acid methionine is drastically elevated in GBMs compared to normal cells, however, it is not known how this methionine is utilized or whether it relates to GBM treatment resistance.Here, we find that radiation acutely increases the levels of methionine-related metabolites in a variety of treatment-resistant GBM models. Stable isotope tracing studies further revealed that radiation acutely activates methionine to S-adenosyl methionine (SAM) conversion through an active signaling event mediated by the kinases of the DNA damage response. In vivo tumor SAM synthesis increases after radiation, while normal brain SAM production remains unchanged, indicating a tumor-specific metabolic alteration to radiation. Pharmacological and dietary strategies to block methionine to SAM conversion slowed DNA damage response and increased cell death following radiation in vitro. Mechanistically, these effects are due to depletion of DNA repair proteins and are reversed by SAM supplementation. These effects are selective to GBMs lacking the methionine salvage enzyme methylthioadenosine phosphorylase. Pharmacological inhibition of SAM synthesis hindered tumor growth in flank and orthotopic in vivo GBM models when combined with radiation. By contrast, methionine depletion does not reduce tumor SAM levels and fails to radiosensitize intracranial models, indicating depleting SAM, as opposed to simply lowering methionine, is critical for hindering tumor growth in intracranial models of GBM. These results highlight a new signaling link between DNA damage and SAM synthesis and define the metabolic fates of methionine in GBM in vivo. Inhibiting radiation-induced SAM synthesis slows DNA repair and augments radiation efficacy in GBM. Using MAT2A inhibitors to deplete SAM may selectively overcome treatment resistance in GBMs with defective methionine salvage while sparing normal brain.

PATH-34. IT NEVER RAINS BUT IT POURS - DIAGNOSING ANAPLASTIC PLEOMORPHIC XANTHASTROCYTOMA TWICE IN A FORTNIGHT - OUR EXPERIENCE IN A RESOURCE CONSTRAINED SETTING

Abstract Grade 3 Pleomorphic Xanthastrocytoma is a rare tumour (less than 1% of all astrocytic glial neoplasms). Present most commonly with seizures. Surgery and Radiation remain the mainstay of treatment with no clear role for traditional systemic chemotherapies; BRAF inhibitors show some benefit. We report two cases who were diagnosed within a month of each other - one was initially diagnosed in 2017- presented with altered smell, headache and diplopia. Found to have a well-defined lobulated heterogeneously enhancing suprasellar parasellar lesion. After Gross Total Resection, was diagnosed from various centers as Anaplastic Ependymoma WHO Grade III; Gllioblastoma WHO Grade IV; Anaplastic Pleomorphic Xanthastrocytoma WHO Grade III. Based on immunohistochemistry, was considered Anaplastic Ependymoma WHO Grade III. Spine screening was negative; received adjuvant Radiation 60Gy in 30 fractions. After a disease free interval of 6 years, developed a recurrence at the post op site. He underwent repeat decompression and the histopathology showed Grade 3 Pleomorphic Xanthastrocytoma. Immunohistochemistry- OLIG2: diffuse positive; R132H p.IDH1: negative; ATRX: intact nuclear expression; Mismatch repair proteins (MSH2, MSH6, MLH1, PMS2); intact nuclear expression; MIB-1 labelling index: 10-12%; H3 p.K27M: negative; GFAP: variable positivity; Podoplanin and p53 shows focal positivity; EMA, CD 34 are negative. He competed Reirradiation in January 2024. He is asymptomatic and on follow up. The second case -15 year old boy presented with episodes of absence seizures. Imaging showed a predominantly extraaxial lesion at tentorial apex. He underwent decompression and histopathology showed Grade 3 Pleomorphic Xanthastrocytoma. Immunohistochemistry - cells positive for S100 and variably positive for GFAP, OLIG2 and vimentin; ATRX: Retained expression, IDH1p.R132H, EMA, Synaptophysin, Neu N: Negative, p53: Focally positive, CD34: Positive in few tumour cells, Ki-67: 14-16 %.V600 mutation detected in the BRAF gene. Had Adjuvant RT 60 Gy in 30 fractions -completed in March 2024. MRI at 1 month - no significant change in overall tumour burden compared to post operative MRI. Was offered Dabrafenib with Trametinib.

The Transcriptomic Response of Cells of the Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation

As areas of application of terahertz (THz) radiation expand in science and practice, evidence is accumulating that this type of radiation can affect not only biological molecules directly, but also cellular processes as a whole. In this study, the transcriptome in cells of the thermophilic bacterium Geobacillus icigianus was analyzed immediately after THz irradiation (0.23 W/cm2, 130 μm, 15 min) and at 10 min after its completion. THz irradiation does not affect the activity of heat shock protein genes and diminishes the activity of genes whose products are involved in peptidoglycan recycling, participate in redox reactions, and protect DNA and proteins from damage, including genes of chaperone protein ClpB and of DNA repair protein RadA, as well as genes of catalase and kinase McsB. Gene systems responsible for the homeostasis of transition metals (copper, iron, and zinc) proved to be the most sensitive to THz irradiation; downregulation of these systems increased significantly 10 min after the end of the irradiation. It was also hypothesized that some negative effects of THz radiation on metabolism in G. icigianus cells are related to disturbances in activities of gene systems controlled by metal-sensitive transcription factors.

The synergy between alkylating agents and ERCC1-XPF inhibitors is p53 dependent.

DNA repair plays a major role in maintaining genomic stability, thus limiting the transformation of normal cells into cancer cells. However, in cancer patients treated with DNA-targeting drugs, DNA repair can decrease efficacy by removing the damage generated by such molecules that is needed to induce pharmacological activity. Inhibiting DNA repair thus represents an interesting approach to potentiating the activity of chemotherapy in this setting. Here, we continue the characterization of an inhibitor of the interaction between Excision Repair Cross-Complementing Rrodent repair deficiency complementation group 1 (ERCC1) and Xeroderma Pigmentousum group F (XPF) (B9), two key proteins of nucleotide excision repair. We used various cell lines and co-incubation studies for the determination of cell survival and DNA repair capacities. We show that it is synergistic with other platinum derivatives than previously described, and that synergy is lacking in cells not expressing ERCC1 or XPF. Finally, a series of experiments show that potentiation is observed only in cells expressing wild-type p53. Our results confirm the mechanism of action of our ERCC1-XPF inhibitor and give important additional data on this approach to enhance the activity of already existing cancer drugs.

EZH1/2 plays critical roles in oocyte meiosis prophase I in mice

Backgroudabnormalities or defects in oocyte meiosis can result in decreased oocyte quality, reduced ovarian reserve, and female diseases. However, the mechanisms of oocyte meiosis remain largely unknown, especially epigenetic regulation. Here, we explored the role of EZH1/2 (histone methyltransferase of H3K27) in mouse oocyte meiosis by inhibiting its activity and deleting its gene.Resultswith embryonic ovary cultured in vitro, EZH1/2 was demonstrated to be essential for oocyte development during meiosis prophase I in mice. Activity inhibition or gene knockout of EZH1/2 resulted in cell apoptosis and a reduction in oocyte numbers within embryonic ovaries. By observing the expression of some meiotic marker protein (γ-H2AX, diplotene stage marker MSY2 and synapsis complex protein SCP1), we found that function deficiency of EZH1/2 resulted in failure of DNA double-strand breaks (DSBs) repair and break of meiotic progression in fetal mouse ovaries. Moreover, Ezh1/2 deficiency led to the suppression of ATM (Ataxia Telangiectasia Mutated kinase) phosphorylation and a decrease in the expression of key DNA repair proteins Hormad1, Mre11, Rad50, and Nbs1 in fetal mouse ovaries, underscoring the enzyme’s pivotal role in initiating DNA repair. RNA-seq analysis revealed that Ezh1/2-deletion induced abnormal expression of multiple genes involved into several function of oocyte development in embryonic ovaries. Knockout of Ezh1/2 in ovaries also affected the levels of H3K9me3 and H4K20me2, as well as the expression of their target genes L3mbtl4 and Fbxo44.Conclusionsour study demonstrated that EZH1/2 plays a role in the DSBs repair in oocyte meiosis prophase I via multiple mechanisms and offers new insights into the physiological regulatory role of histone modification in fetal oocyte guardianship and female fertility.

PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

Efficient DNA double strand break (DSB) repair is necessary for genomic stability and determines efficacy of DNA damaging cancer therapeutics. Spatiotemporal dynamics and post-translational modifications of repair proteins at DSBs dictate repair efficacy. Here, we identified a non-canonical function of GCN5 in regulating both HR and NHEJ repair post genotoxic stress. Mechanistically, genotoxic stress induced GCN5 recruitment to DSBs. GCN5 PARylation by PARP1 was essential for its recruitment, acetyltransferase activity and DSB repair function. Liquid chromatography-mass spectrometry (LC–MS) identified DNA-PKcs as part of GCN5 interactome. In-vitro acetyltransferase assays revealed that GCN5 acetylates DNA-PKcs at K3241 residue, a prerequisite for DNA-PKcs S2056 phosphorylation and DSB recruitment. Alongside, ChIP-qPCR revealed GCN5 mediates transcription of PRKDC via H3K27Ac acetylation in its promoter region (− 710 to − 554). Genetic perturbation of GCN5 also decreased CHEK1, NBN1, TP53BP1, POL-L transcription and abrogated ATM, BRCA1 activation. Accordingly, GCN5 loss led to persistent ɣ-H2AX foci formation, compromised in-vivo HR-NHEJ and caused GBM radio-sensitization. Importantly, PARP1 inhibition phenocopied GCN5 loss. Together, this study identifies an untraversed DSB repair function of GCN5 and provides mechanistic insights into transcriptional as well as post-translational regulation of pivotal HR-NHEJ factors. Alongside, it highlights the translational importance of PARP1-GCN5 axis in mediating GBM radio-resistance.

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Excellent concordance of the molecular classification between preoperative biopsy and final hysterectomy in endometrial carcinoma

ObjectiveThe 2023 International Federation of Gynecology and Obstetrics classification with molecular classification shows superior discriminatory ability compared to staging systems lacking molecular data. However, the accuracy of endometrial biopsy data in molecular classification remains uncertain. This study aimed to assess the concordance of molecular classifications between preoperative biopsy and hysterectomy to predict prognosis before surgical staging. MethodsEndometrial biopsies and corresponding hysterectomy specimens were collected at the National Cancer Center Hospital between 2012 and 2023. Immunohistochemistry for p53 and mismatch repair (MMR) proteins and next-generation sequencing of all exons of polymerase epsilon (POLE) were performed. Given the limited number of POLE mut cases in prior studies, we prepared a POLE mut-enriched cohort. Cohen's kappa estimates were used to determine concordance for molecular and clinicopathological subgroup assignments. ResultsAmong 70 patients classified into four molecular subtype groups, 33 exhibited POLE mutations, 15 showed loss of MMR protein expression, 13 had p53-abnormality, and 9 had no specific molecular profile. Concordance between biopsy and hysterectomy specimens was 100% (κ = 1.000). In contrast, histological types and grades between biopsy and surgical specimens showed moderate and substantial agreement (κ = 0.420 and κ = 0.780, respectively). ConclusionsMolecular subtypes were completely consistent with those derived from surgical specimens, demonstrating high concordance between preoperative and postoperative molecular classifications. This suggests that endometrial biopsies could reliably predict prognosis. Future studies should investigate how biopsy-based molecular profiling influences treatment planning and patient outcomes.

A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function.

Cockayne syndrome (CS) is a premature ageing condition characterized by microcephaly, growth failure, and neurodegeneration. It is caused by mutations in ERCC6 or ERCC8 encoding for Cockayne syndrome B (CSB) and A (CSA) proteins, respectively. CSA and CSB have well-characterized roles in transcription-coupled nucleotide excision repair, responsible for removing bulky DNA lesions, including those caused by UV irradiation. Here, we report that CSA dysfunction causes defects in the nuclear envelope (NE) integrity. NE dysfunction is characteristic of progeroid disorders caused by a mutation in NE proteins, such as Hutchinson-Gilford progeria syndrome. However, it has never been reported in Cockayne syndrome. We observed CSA dysfunction affected LEMD2 incorporation at the NE and increased actin stress fibers that contributed to enhanced mechanical stress to the NE. Altogether, these led to NE abnormalities associated with the activation of the cGAS/STING pathway. Targeting the linker of the nucleoskeleton and cytoskeleton complex was sufficient to rescue these phenotypes. This work reveals NE dysfunction in a progeroid syndrome caused by mutations in a DNA damage repair protein, reinforcing the connection between NE deregulation and ageing.

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

Intact DNA repair in differentiated cardiomyocytes is essential for maintaining cardiac function in response to pressure overload in mice

Abstract Introduction DNA in every cell is continuously damaged and DNA repair mechanisms are essential for protection against DNA damage-induced aging-related diseases. For example, deficient repair of endogenously generated DNA damage in mice with cardiomyocyte-restricted inactivation of Xpg, is associated with progressive heart failure. Purpose Here we tested the hypothesis that unrepaired DNA damage in differentiated cardiomyocytes increases cardiac vulnerability in response to hemodynamic overload. Methods To increase the burden of spontaneous DNA damage, we generated mice with cardiomyocyte-restricted inactivation of DNA repair endonuclease XPG. At 8 weeks of age, αMHC-Xpgc/- and control (Ctrl) mice were subjected to pressure overload by transverse aortic constriction (TAC). Eight weeks after TAC, left ventricular (LV) function was assessed using echocardiography and hemodynamic measurements, followed by molecular and histological analyses. Results Cardiomyocyte-restricted inactivation of Xpg resulted in systolic as well as diastolic LV dysfunction (Table). TAC-induced LV hypertrophy is similar in both groups (Ctrl 38%; αMHC-Xpgc/- 34%). In Ctrl mice, LV hypertrophy was accompanied by minimal LV dilation and modest changes in systolic and diastolic LV function. Conversely, TAC in αMHC-Xpgc/- produced severe LV dysfunction and resulted in overt congestive heart failure, demonstrated by aggravation of LV dilation, and marked increases in LV end-diastolic pressure, left atrial weight and lung fluid weight, which was accompanied by further increases in the expression levels of the hypertrophic marker genes atrial natriuretic peptide and beta-myosin heavy chain (Table). Moreover, lectin staining revealed a decrease in capillary density and TUNEL staining revealed further elevated levels of myocardial apoptosis. In addition, a significant increase of myocardial collagen content was observed. Conclusion Cardiomyocyte-restricted loss of DNA repair protein XPG increases cardiac vulnerability to develop heart failure in response to pressure overload. These findings underscore the importance of genomic stability for maintenance of cardiac function, not only during basal conditions, but also in response to a pathological stimulus.

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions.

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

Comparative Analysis of Plasma Protein Dynamics in Women with ST-Elevation Myocardial Infarction and Takotsubo Syndrome.

ST-elevation myocardial infarction (STEMI) and Takotsubo syndrome (TS) are two distinct cardiac conditions that both result in sudden loss of cardiac dysfunction and that are difficult to distinguish clinically. This study compared plasma protein changes in 24 women with STEMI and 12 women with TS in the acute phase (days 0-3 post symptom onset) and the stabilization phase (days 7, 14, and 30) to examine the molecular differences between these conditions. Plasma proteins from STEMI and TS patients were extracted during the acute and stabilization phases and analyzed via quantitative proteomics. Differential expression and functional significance were assessed. Data are accessible on ProteomeXchange, ID PXD051367. During the acute phase, STEMI patients showed higher levels of myocardial inflammation and tissue damage proteins compared to TS patients, along with reduced tissue repair and anti-inflammatory proteins. In the stabilization phase, STEMI patients exhibited ongoing inflammation and disrupted lipid metabolism. Notably, ADIPOQ was consistently downregulated in STEMI patients in both phases. When comparing the acute to the stabilization phase, STEMI patients showed increased inflammatory proteins and decreased structural proteins. Conversely, TS patients showed increased proteins involved in inflammation and the regulatory response to counter excessive inflammation. Consistent protein changes between the acute and stabilization phases in both conditions, such as SAA2, CRP, SAA1, LBP, FGL1, AGT, MAN1A1, APOA4, COMP, and PCOLCE, suggest shared underlying pathophysiological mechanisms. This study presents protein changes in women with STEMI or TS and identifies ADIPOQ, SAA2, CRP, SAA1, LBP, FGL1, AGT, MAN1A1, APOA4, COMP, and PCOLCE as candidates for further exploration in both therapeutic and diagnostic contexts.

Non-classical action of Ku70 promotes Treg suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma.

Ku70, a DNA repair protein, binds to the damaged DNA ends and orchestrates the recruitment of other proteins to facilitate repair of DNA double-strand breaks. Besides its essential role in DNA repair, several studies have highlighted non-classical functions of Ku70 in cellular processes. However, its function in immune homeostasis and anti-tumor immunity remains unknown. Here, we discovered a marked association between elevated Ku70 expression and unfavorable prognosis in lung adenocarcinoma, focusing specifically on increased Ku70 levels in tumor-infiltrated Treg cells. Using a lung-colonizing tumor model of in mice with Treg-specific Ku70 deficiency, we demonstrated that deletion of Ku70 in Treg cells led to a stronger anti-tumor response and slower tumor growth due to impaired immune-suppressive capacity of Treg cells. Furthermore, we confirmed that Ku70 played a critical role in sustaining the suppressive function of human Treg cells. We found that Ku70 bound to FOXP3 and occupied FOXP3-bound genomic sites to support its transcriptional activities. These findings not only unveil a non-homologous end joining (NHEJ)-independent role of Ku70 crucial for Treg suppressive function, but also underscore the potential of targeting Ku70 as an effective strategy in cancer therapy, aiming to both restrain cancer cells and enhance pulmonary anti-tumor immunity.

Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence

DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes.