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Srinivasan, R.

Publications and source records attributed to Srinivasan, R..

At least 37 records · Page 2Linked to original sources

Evaluation of enzyme activity predictions for variants of unknown significance in Arylsulfatase A

Continued advances in variant effect prediction are necessary to demonstrate the ability of machine learning methods to accurately determine the clinical impact of variants of unknown significance (VUS). Towards this goal, the ARSA Critical Assessment of Genome Interpretation (CAGI) challenge was designed to characterize progress by utilizing 219 experimentally assayed missense VUS in the Arylsulfa-tase A (ARSA) gene to assess the performance of community-submitted predictions of variant functional effects. The challenge involved 15 teams, and evaluated additional predictions from established and recently released models. Notably, a model developed by participants of a genetics and coding bootcamp, trained with standard machine-learning tools in Python, demonstrated superior performance among sub-missions. Furthermore, the study observed that state-of-the-art deep learning methods provided small but statistically significant improvement in predictive performance compared to less elaborate techniques. These findings underscore the utility of variant effect prediction, and the potential for models trained with modest resources to accurately classify VUS in genetic and clinical research.

genetics↗

The smoking cessation drug cytisine requires systemically circulating estrogen for sex-specific neuroprotection in female parkinsonian mice

The smoking cessation drug cytisine exerts neuroprotection in substantia nigra pars compacta (SNc) dopaminergic (DA) neurons of female but not male 6-hydroxydopamine (6-OHDA) lesioned parkinsonian mice. To address the important question of whether circulating estrogen mediates this effect, we employ two mouse models aimed at depleting systemically circulating estrogen: (i) bilateral ovariectomy (OVX), and (ii) aromatase inhibition with systemically administered letrozole. In both models, depleting systemically circulating estrogen in female 6-OHDA lesioned parkinsonian mice results in the loss of cytisine-mediated neuroprotection as measured using apomorphine-induced contralateral rotations and SNc DA neurodegeneration. Our experiments also reveal that OVX alone exerts neuroprotection in SNc DA neurons due to compensatory changes not observed in the letrozole model, which underscores the importance of using independent models of estrogen depletion to study neuroprotection. Taken together, our findings suggest that the smoking cessation drug cytisine is a viable neuroprotective drug for pre-menopausal women with Parkinsons disease.

neuroscience↗

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

cancer biology↗

Identifying Factors Important for Conservation at Sites of Synonymous Variations

Synonymous mutations can have a deleterious effect leading to disease, even though they are not protein altering. Variations at genomic sites leading to synonymous variants are frequently highly conserved across species. Several prediction methods have been developed to assess the impact of synonymous mutations and are highly dependent on having validated sets of both deleterious and benign synonymous mutations. However, validated data available for deleterious synonymous mutations is sparse unlike for missense mutations. Rather than develop a model for predicting pathogenicity of synonymous variants, we seek to understand the relative importance of various factors that lead to conservation at sites of synonymous variants. Our study built machine learning models using various features on a large set of reported and generated synonymous variants (Zeng Z et al, 2019) to predict conservation (Genomic Evolutionary Rate Profiling - Rejected Substitution (GERP RS) base scores and Phylogenetic p-values for 100 vertebrates (PP100)) at genomic sites. We used the extreme gradient boosting classifier to classify sites as high, medium and low conservation at different cutoffs. Our experiments report an AUC between 0.74-0.79 and the sensitivity was significant. Of the features we explored, a few alternate allele independent properties were repeatedly flagged as having high impact. These findings provide information for predictors to further improve models for synonymous variant impact.

bioinformatics↗

Impaired embryo sac cellularization by PMEI gene mutation affects gamete specification and twin plants in Arabidopsis

Arabidopsis lines with loss-of-function mutations in the gene encoding Embryo sac Pectin MethylEsterase Inhibitor (Atepmei) were found to have short silique and high seed sterility. Examination of tissue-cleared mature ovules (FG7-stage) revealed irregularly positioned nuclei within the embryo sacs. Instead of horse-shoe-shaped ovules, defective globular ovules without proper micropylar and chalazal ends were found. Embryo sac cell-type-specific GFP marker expression studies confirmed gamete and accessory cell identity alterations. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs, possibly due to defect in embryo sac cellularization. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of Ruthenium red and tannic acid-ferric chloride staining of Atepmei mutant developing ovules, conferred its interaction with the specific PME in proper cell wall formation and maintenance around embryo sac nuclei which also coincide with its fate as a specific gamete. This is the first report implicating role of cell wall in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

plant biology↗

GluK1 kainate receptors are necessary for functional maturation of parvalbumin interneurons regulating amygdala circuit function

Parvalbumin expressing interneurons (PV INs) are key players in the local inhibitory circuits and their developmental maturation coincides with the onset of adult-type network dynamics in the brain. Glutamatergic signaling regulates emergence of the unique PV IN phenotype, yet the receptor mechanisms involved are not fully understood. Here we show that GluK1 subunit containing kainate receptors (KARs) are necessary for development and maintenance of the neurochemical and functional properties of PV INs in the basolateral amygdala (BLA). Ablation of GluK1 expression specifically from PV INs resulted in low parvalbumin expression and loss of characteristic high firing rate throughout development. In addition, we observed reduced spontaneous excitatory synaptic activity at adult GluK1 lacking PV INs. Intriguingly, inactivation of GluK1 expression in adult PV INs was sufficient to abolish the PV phenotype, suggesting a role for GluK1 in dynamic regulation of PV IN maturation state. The PV IN dysfunction in the absence of GluK1 perturbed feedforward inhibition and long-term potentiation (LTP) in the BLA and resulted in developmentally originating changes in the glutamatergic connectivity to BLA principal neurons. Behaviorally, the absence of GluK1 from PV INs associated with hyperactivity and increased fear of novelty. These results indicate a critical role for GluK1 KARs in regulation of PV IN function across development and suggest GluK1 as a potential therapeutic target for pathologies involving PV IN malfunction.

neuroscience↗

An amphipathic helix facilitates direct membrane binding of Mycoplasma FtsZ

Cell division in bacteria is initiated by constriction of the Z-ring comprising two essential proteins FtsZ and FtsA. Despite our knowledge about the crucial function of the Z-ring in bacterial division, the precise roles and mechanism of how FtsZ and FtsA drive cell constriction remain elusive. FtsZ/FtsA in wall-less bacteria like mycoplasmas is an ideal model system for obtaining mechanistic insights into Z-ring constriction in the absence of cell wall machinery. In this study, we have analyzed FtsZ and FtsA sequences of 113 mycoplasma species and compared with the corresponding protein sequences in cell-walled bacteria. We report a phylogenetically distinct group of 12 species that possess FtsZs without the canonical FtsA interacting conserved C-terminal peptide (CCTP) motif. Interestingly, these FtsZs contain a putative membrane-binding amphipathic helix as an N-terminal or C-terminal extension to the globular FtsZ domain. As a proof-of-concept, we experimentally show that the proposed C-terminal amphipathic helix in M. genitalium FtsZ binds liposomes in vitro as well as localizes to E. coli membrane in vivo. Additionally, we identify a putative cholesterol recognition motif within the C-terminal amphipathic helix region of M. genitalium FtsZ. Our study catalogues the functional variations of membrane attachment by the FtsZ and FtsA system in cell wall-less mycoplasmas and provides a new perspective to study novel functions of FtsZ/A system in cell division. ImportanceZ-ring and peptidoglycan synthesis machinery both play crucial roles in bacterial cell division. Currently, our knowledge about how FtsZ and FtsA, the two primary components of the Z-ring, function, is limited to cell-walled bacteria where ring constriction is coupled to peptidoglycan synthesis. Cell wall-less bacterial FtsZ/A system is an excellent model to study the mechanism of Z-ring constriction in the absence of cell wall synthesis machinery. Here, we analysed FtsZ protein sequences across mycoplasma species and identified their characteristic sequence features. Our study reveals a novel group of FtsZs from mycoplasma with an inherent membrane binding and probable cholesterol sensing amphipathic motif, which serves as a new paradigm to explore fundamental roles of FtsZ and FtsA in Z-ring constriction during bacterial division.

microbiology↗

A cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase interacts with begomovirus capsid proteins and modulates virus retention within its vector

Begomoviruses are whitefly-transmitted ss-DNA viruses infecting dicotyledonous plants and contribute to major economic losses to global crop production. Invasion and establishment of an aggressive species of B. tabaci, known as the B cryptic species, has severely constrained vegetable production in the southeastern and southwestern United States. Disruption of genes/pathways critical for whitefly mediated transmission can be effective for the management of begomoviruses. In this study, yeast two hybrid (Y2H)-based screening of B. tabaci cDNA library identified a cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE4) of the whitefly as an interacting partner with capsid proteins (CPs) of old- and new-world begomoviruses. Interactions of PDE4 with begomovirus CPs were validated by GST-pull-down assays, co-immunoprecipitation, and co-immunolocalization in whitefly midgut. The PDE4 family of enzymes hydrolyze cAMP and regulate intracellular cAMP levels. This study revealed that elevation of cAMP within whitefly by chemically inhibiting or gene (PDE4) silencing resulted in increased retention and transmission of begomoviruses. Similarly, decreased cAMP levels resulted in reduced begomovirus retention. The results of this study demonstrate that whitefly mediated transmission of begomoviruses is regulated by intracellular cAMP by unknown mechanisms. ImportanceBegomoviruses, transmitted by the sweetpotato whitefly, (Bemisa tabaci Gennadius), are the causal agents of many economically important plant virus diseases. Lack of host plant resistance against begomoviruses, high whitefly abundance, and whiteflys ability to develop insecticide resistance rapidly often renders commonly used management practice ineffective. This study demonstrates how begomovirus retention within whitefly and its transmission can be modulated by altering cAMP expression of its insect vector. Naturally occurring bio-pesticides that target insect cAMPs are known. Our findings can lead to alternative strategies for the management of begomoviruses by targeting whitefly cAMP using chemicals, botanicals, or RNAi-based insecticides.

molecular biology↗

Selective inhibitors of the Aurora A-TPX2 protein-protein interaction exhibit in vivo efficacy as targeted anti-mitotic agents

The protein kinase Aurora A, and its close relative, Aurora B, regulate human cell division. Aurora A is frequently overexpressed in cancers of the breast, ovary, pancreas and blood, provoking genome instability and resistance to anti-mitotic chemotherapy. Intracellular localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. Here, we have used fragment-based, structure-guided lead discovery to develop small-molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI). These compounds act by novel mechanism compared to existing Aurora A inhibitors and they are highly specific to Aurora A over Aurora B. Our biophysically, structurally and phenotypically validated lead compound, CAM2602, exhibits oral bioavailability, favourable pharmacokinetics, pharmacodynamic biomarker modulation, and arrest of growth in tumour xenografts. Consistent with our original finding that Aurora A overexpression drives taxane-resistance in cancer cells, inhibition of Aurora A-TPX2 PPI synergizes with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.

biochemistry↗

Constitutive protein degradation induces acute cell death via proteolysis products

Modulation of proteolysis is an emerging therapeutic mainstay. The clinical success of thalidomide and analogs has inspired development of rationally-designed therapeutics that repurpose endogenous degradation machinery to target pathogenic proteins. However, it is unknown whether target removal is the critical effect that drives degrader-induced efficacy. Here we report that proteasome-generated peptides actively initiate degrader-induced cell death. Utilizing BET family degraders as exemplars, we find that induced proteasomal degradation of the BRD4-long isoform (BRD4-L) generates neo-amino-terminal peptides that neutralize Inhibitor of Apoptosis (IAP) proteins to precipitate cell death. Depletion of BRD4-L paradoxically suppresses caspase activation induced by numerous BET degraders. An unbiased screen revealed that other degrader compounds, including clinical CELMoDs, rely on the same mechanism to potentiate caspase activation and apoptosis. Finally, in the context of constitutive immunoglobulin proteostasis within multiple myeloma cells, we report that therapeutic proteasomal protease inhibition alters the peptide repertoire to neutralize IAPs, thus contributing to the clinical efficacy of bortezomib. Together, these findings clarify the counterintuitive clinical benefit achieved by combining thalidomide analogs with proteasome inhibitors. Our study reveals a previously unrealized pro-apoptotic function of the peptides generated by a variety of proteolysis-modulating compounds, that provide design considerations to maximize therapeutic benefit.

cell biology↗

MAGE: Strain Level Profiling of MetagenomeSamples

Metagenomic profiling from sequencing data aims to disentangle a microbial sample at lower ranks of taxonomy, such as species and strains. Deep taxonomic profiling involving accurate estimation of strain level abundances aids in precise quantification of the microbial composition, which plays a crucial role in various downstream analyses. Existing tools primarily focus on strain/subspecies identification and limit abundance estimation to the species level. Abundance quantification of the identified strains is challenging and remains largely unaddressed by the existing approaches. We propose a novel algorithm MAGE (Microbial Abundance GaugE), for accurately identifying constituent strains and quantifying strain level relative abundances. For accurate profiling, MAGE uses read mapping information and performs a novel local searchbased profiling guided by a constrained optimization based on maximum likelihood estimation. Unlike the existing approaches that often rely on strain-specific markers and homology information for deep profiling, MAGE works solely with read mapping information, which is the set of target strains from the reference collection for each mapped read. As part of MAGE, we provide an alignment-free and kmer-based read mapper that uses a compact and comprehensive index constructed using FM-index and R-index. We use a variety of evaluation metrics for validating abundances estimation quality. We performed several experiments using a variety of datasets, and MAGE exhibited superior performance compared to the existing tools on a wide range of performance metrics.

bioinformatics↗

Improving classification and reconstruction of imagined images from EEG signals

Decoding brain activity related to specific tasks, such as imagining something, is important for brain computer interface (BCI) control. While decoding of brain signals, such as functional magnetic resonance imaging (fMRI) signals and electroencephalography (EEG) signals, during observing visual images and while imagining images has been previously reported, further development of methods for improving training, performance, and interpretation of brain data was the goal of this study. We applied a Sinc-EEGNet to decode brain activity during perception and imagination of EEG stimuli, and added an attention module to extract the importance of each electrode or frequency band. We also reconstructed images from brain activity by using a generative adversarial network (GAN). By combining the EEG recorded during a visual task (perception) and imagination task, we have successfully boosted the accuracy of classifying EEG data in the imagination task and improved the quality of reconstruction by GAN. Our result indicates that the brain activity evoked during the visual task is present in the imagination task and can be used for better classification of the imagined image. By using the attention module, we can derive the spatial weights in each frequency band and contrast spatial or frequency importance between tasks from our model. Imagination tasks are classified by low frequency EEG signals over temporal cortex, while perception tasks are classified by high frequency EEG signals over occipital and frontal cortex. Combining data sets in training results in a balanced model improving classification of the imagination task without significantly changing performance in the visual task. Our approach not only improves performance and interpretability but also potentially reduces the burden on training since we can improve the accuracy of classifying a relatively hard task with high variability (imagination) by combining with the data of the relatively easy task, observing visual images.

neuroscience↗

Dimensionality reduction of calcium-imaged neuronal population activity

Calcium imaging has been widely adopted for its ability to record from large neuronal populations. To summarize the time course of neural activity, dimensionality reduction methods, which have been applied extensively to population spiking activity, may be particularly useful. However, it is unclear if the dimensionality reduction methods applied to spiking activity are appropriate for calcium imaging. We thus carried out a systematic study of design choices based on standard dimensionality reduction methods. We also developed a novel method to perform deconvolution and dimensionality reduction simultaneously (termed CILDS). CILDS most accurately recovered the single-trial, low-dimensional time courses from calcium imaging that would have been recovered from spiking activity. CILDS also outperformed the other methods on calcium imaging recordings from larval zebrafish and mice. More broadly, this study represents a foundation for summarizing calcium imaging recordings of large neuronal populations using dimensionality reduction in diverse experimental settings.

neuroscience↗

Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases risk for neurodegeneration. Among the various cells participating in BBB and NVU function, spontaneous Ca2+ signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with changes in spontaneous Ca2+ signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed a genetically encoded Ca2+ indicator, Lck-GCaMP6f in DLS astrocytes of young (3-4 month) and aging (20-24 month) mice. Compared to young mice, endfeet in the DLS of aging mice demonstrated a decrease in calreticulin (CALR) expression, and dramatic alterations in the dynamics of endfoot membrane-associated and mitochondrial Ca2+ signals. While young mice required both extracellular and endoplasmic reticulum (ER) Ca2+ sources for generating endfoot Ca2+ signals, aging mice showed exclusive dependence on ER Ca2+. These data suggest that aging is associated with significant changes in Ca2+ buffers and Ca2+ signals within astrocytic endfeet, which has important implications for understanding mechanisms involved in aging-related impairment of the BBB and NVU. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/461710v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d61571org.highwire.dtl.DTLVardef@19e2f9org.highwire.dtl.DTLVardef@18455cborg.highwire.dtl.DTLVardef@11ca928_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAging mice show reduced calreticulin expression in astrocytic endfeet C_LIO_LIAging astrocytic endfeet show dramatic changes in spontaneous Ca2+ activity C_LIO_LICa2+ signals in aging endfeet depend exclusively on endoplasmic reticulum Ca2+ C_LI

neuroscience↗

Detecting the state of drowsiness induced by propofol by spatial filter and machine learning algorithm on EEG recording

To accurately measure the depth of anesthesia has been a challenge for both anesthesiologists and engineers who work on developing tools of measurements. This study aims to use a machine-learning algorithm to predict the drowsy state, a transitional depth of sedation during propofol anesthesia. The data used in this study were scalp EEG (electroencephalogram) recordings selected from the University of Cambridge Repository. Raw EEG recordings were preprocessed into power spectrum matrices one second per sample. A total of 170 samples (110 awake samples and 60 drowsy samples) were used. A CNN (Convolutional Neural Network) for the MNIST (Modified National Institute of Standards and Technology) dataset was applied on these EEG power spectrum matrices. Due to the small dataset volume, Leave-One-Out cross-validation was used to train the data. Results of the training accuracy reached 99.69%. And test accuracy averaged 96.47%. Overall, the model is able to predict the state of drowsiness during propofol anesthesia. This provides the potential to develop EEG monitoring devices with closed-loop feedback of such a machine learning algorithm that controls the titration of the dosage of anesthetic administration and the depth of anesthesia.

neuroscience↗

Filament dynamics driven by ATP hydrolysis modulates membrane binding of the bacterial actin MreB

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. While the eukaryotic actin utilizes ATP hydrolysis to drive filament treadmilling, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we report mechanistic insights into the interplay between nucleotide-binding, ATP hydrolysis and membrane-binding of Spiroplasma citri MreB5 (ScMreB5). Antiparallel double protofilament assembly of ScMreB5WT with ATP, ADP or AMPPNP and an ATPase deficient mutant ScMreB5E134A demonstrate that the filaments assemble independent of ATP hydrolysis. However, capture of the filament dynamics revealed that efficient filament formation, bundling through lateral interactions and filament disassembly are affected in ScMreB5E134A. Hence, the catalytic glutamate (Glu134 in ScMreB5) plays a dual role - it functions as a switch by sensing the ATP-bound state for filament assembly, and by assisting hydrolysis for triggering disassembly. Glu134 mutation also exhibits an allosteric effect on membrane binding, as observed from the reduced liposome binding compared to that of the wild type. Thus, ATP hydrolysis can modulate filament length and bundling, and consequently the orientation of MreB filaments on the cell membrane depending on the curvature. Binding of ScMreB5 with liposomes is mediated by surface charge-based interactions, demonstrating paralog and organism specific features for MreB function. We conclude that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis has been repurposed for modulating curvature-dependent organization of filaments on the membrane.

biochemistry↗

Extracellular S100B alters spontaneous Ca2+ fluxes in dopaminergic neurons via L-type voltage gated calcium channels: Implications for Parkinson's disease

Parkinsons disease (PD) is associated with an abnormal increase in S100B within the midbrain and cerebrospinal fluid. In addition, overexpression of S100B in mice accelerates the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons, suggesting a role for this protein in PD pathogenesis. We found that in the mouse SNc, S100B labeled astrocytic processes completely envelop the somata of tyrosine hydroxylase (TH) positive DA neurons. Based on this finding, we rationalized that abnormal increases in extracellularly secreted S100B by astrocytic processes in the SNc could alter DA neuron activity, thereby causing dysregulated midbrain function. To test this hypothesis, we measured the effect of bath perfused S100B peptide on the frequency and amplitude of spontaneous calcium fluxes in identified TH+ and TH- midbrain neurons from 3-week old mouse primary midbrain cultures. Acute exposure to 50 pM S100B caused a 2-fold increase in calcium flux frequency only in TH+ DA neurons. The L-type voltage gated calcium channel (VGCC) inhibitor, diltiazem eliminated S100B-mediated increases in DA neuron calcium flux frequency, while the T-type specific VGCC blocker mibefradil failed to inhibit the stimulatory effect of S100B. Chronic exposure to S100B caused a 3-fold reduction in calcium flux frequencies of TH+ neurons and also reduced calcium flux amplitudes in TH- neurons by [~]4-fold. Together, our results suggest that exposure to S100B pathologically alters spontaneous calcium activity in midbrain neurons via an extracellular mechanism involving L-type VGCCs expressed in DA neurons. These findings are relevant to understanding mechanisms underlying DA neuron loss during PD. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/432751v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@7d3fd3org.highwire.dtl.DTLVardef@e96b3corg.highwire.dtl.DTLVardef@7f6d21org.highwire.dtl.DTLVardef@1d5bde4_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIExtracellular S100B increases Ca2+ fluxes in dopaminergic neurons C_LIO_LIL-type VGCCs in dopaminergic neurons are required for S100B-mediated increases in Ca2+ fluxes C_LIO_LIChronic S100B alters Ca2+ fluxes in dopaminergic and non-dopaminergic neurons C_LI

neuroscience↗

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily-conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted -methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionising radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

cancer biology↗