FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways.

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

USP7 substrates identified by proteomics analysis reveal the specificity of USP7.

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

PAF remodels the DREAM complex to bypass cell quiescence and promote lung tumorigenesis.

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

A Surge of DNA Damage Links Transcriptional Reprogramming and Hematopoietic Deficit in Fanconi Anemia.

Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.

Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis.

Host-virus protein-protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity-labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Further characterization of these interactions and comparison to other large-scale study of cellular responses to SARS-CoV-2 infection elucidated how distinct SARS-CoV-2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID-19. The interactomes of two key SARS-CoV-2-encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS-CoV-2 provides valuable resources for the understanding and treating of this disease.

ATM Creates a veil of transcriptional silence.

The ATM kinase orchestrates diverse responses to DNA damage. By simultaneously monitoring transcription and DNA-damage responses in single cells, Shanbhag et al. (2010) now uncover a role of ATM in preventing transcription near DNA double-strand breaks.

Integrated screens uncover a cell surface tumor suppressor gene KIRREL involved in Hippo pathway.

Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that Kirrel1 loss markedly promoted tumor growth in vivo. Moreover, KIRREL was significantly enriched in a separate CRISPR screen based on a specific Hippo pathway reporter. Further studies revealed that KIRREL binds directly to SAV1 to activate the Hippo tumor suppressor pathway. Together, our integrated screens reveal a cell surface tumor suppressor involved in the Hippo pathway and highlight the potential of these approaches in biomedical research.

Engineering the d-Orbital Energy of Metal-Organic Frameworks-Based Solid-State Electrolytes for Lithium-Metal Batteries.

Having an orbital-level understanding of the relationship between the electronic state of a central metal in metal-organic frameworks (MOFs) as solid-state electrolytes (SSEs) and Li+ ion conductivity is crucial yet challenging for lithium-metal batteries (LMBs). In this study, we report the synthesis of functionalized UiO-66 as a model system to investigate the relationship between the d-band energy of Zr 3d orbitals and Li+ ion conductivity. Specifically, the NO2 group in electron-withdrawing NO2-decorated UiO-66 (NO2-UiO-66) can capture electron from ZrO8 sites, resulting the increased energy in 3dz2 and 3dxz/yz orbitals of Zr atom. The high-energy 3dz2 and 3dxz/yz orbitals of Zr in NO2-UiO-66 hybridize with the 2pz and 2px/y orbitals of O in ClO4-, leading to decreased antibonding orbital energy and resulting in a strong adsorption, ultimately immobilizing the anions and enhancing ion conductivities. Establishing the correlation between the d-orbital energy and Li+ ion conductivity may create a descriptor for designing efficient SSEs for LMBs.

Tuning Two-Dimensional Phthalocyanine Dual Site Metal-Organic Framework Catalysts for the Oxygen Reduction Reaction.

Metal-organic frameworks (MOFs) offer an interesting opportunity for catalysis, particularly for metal-nitrogen-carbon (M-N-C) motifs by providing an organized porous structural pattern and well-defined active sites for the oxygen reduction reaction (ORR), a key need for hydrogen fuel cells and related sustainable energy technologies. In this work, we leverage electrochemical testing with computational models to study the electronic and structural properties in the MOF systems and their relationship to ORR activity and stability based on dual transitional metal centers. The MOFs consist of two M1 metals with amine nodes coordinated to a single M2 metal with a phthalocyanine linker, where M1/M2 = Co, Ni, or Cu. Co-based metal centers, in particular Ni-Co, demonstrate the highest overall activity of all nine tested MOFs. Computationally, we identify the dominance of Co sites, relative higher importance of the M2 site, and the role of layer M1 interactions on the ORR activity. Selectivity measurements indicate that M1 sites of MOFs, particularly Co, exhibit the lowest (<4%), and Ni demonstrates the highest (>46%) two-electron selectivity, in good agreement with computational studies. Direct in situ stability characterization, measuring dissolved metal ions, and calculations, using an alkaline stability metric, confirm that Co is the most stable metal in the MOF, while Cu exhibits notable instability at the M1. Overall, this study reveals how atomistic coupling of electronic and structural properties affects the ORR performance of dual site MOF catalysts and opens new avenues for the tunable design and future development of these systems for practical electrochemical applications.

ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.

Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.

Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor.

The Hippo pathway, which plays a critical role in organ size control and cancer, features numerous WW domain-based protein-protein interactions. However, ~100 WW domains and 2,000 PY motif-containing peptide ligands are found in the human proteome, raising a "WW-PY" binding specificity issue in the Hippo pathway. In this study, we have established the WW domain binding specificity for Hippo pathway components and uncovered a unique amino acid sequence required for it. By using this criterion, we have identified a WW domain-containing protein, STXBP4, as a negative regulator of YAP. Mechanistically, STXBP4 assembles a protein complex comprising α-catenin and a group of Hippo PY motif-containing components/regulators to inhibit YAP, a process that is regulated by actin cytoskeleton tension. Interestingly, STXBP4 is a potential tumor suppressor for human kidney cancer, whose downregulation is correlated with YAP activation in clear cell renal cell carcinoma. Taken together, our study not only elucidates the WW domain binding specificity for the Hippo pathway, but also reveals STXBP4 as a player in actin cytoskeleton tension-mediated Hippo pathway regulation.

STAT5 phosphorylation plus minimal residual disease defines a novel risk classification in adult B-cell acute lymphoblastic leukaemia.

The dysregulation of the Janus family tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) is closely related to acute lymphoblastic leukaemia (ALL), whereas the clinical value of phosphorylated STAT5 (pSTAT5) remains elusive. Herein we performed a prospective study on clinical significance of flow cytometry-based pSTAT5 in adult B-ALL patients. A total of 184 patients were enrolled in the Precision-Classification-Directed-Target-Total-Therapy (PDT)-ALL-2016 cohort between January 2018 and December 2021, and STAT5 phosphorylation was detected by flow cytometry at diagnosis. Based on flow-pSTAT5, the population was classified into pSTAT5low (113/184, 61.1%) and pSTAT5high (71/184, 38.9%). Overall survival (OS) and event-free survival (EFS) were inferior in pSTAT5high patients than in those with pSTAT5low (OS, 44.8% vs. 65.2%, p = 0.004; EFS, 23.5% vs. 52.1%, p < 0.001), which was further confirmed in an external validation cohort. Furthermore, pSTAT5 plus flow-based minimal residual disease (MRD) postinduction defines a novel risk classification as being high risk (HR, pSTAT5high + MRD+), standard risk (SR, pSTAT5low + MRD-) and others as moderate-risk group. Three identified patient subgroups are distinguishable with disparate survival curves (3-year OS rates, 36.5%, 56.7% and 76.3%, p < 0.001), which was confirmed on multivariate analysis (hazard ratio 3.53, p = 0.003). Collectively, our study proposed a novel, simple and flow-based risk classification by integrating pSTAT5 and MRD in favour of risk-guided treatment for B-ALL.

Rationally Construction of Mn-Doped RuO2 Nanofibers for High-Activity and Stable Alkaline Ampere-Level Current Density Overall Water Splitting.

Nowadays, highly active and stable alkaline bifunctional electrocatalysts toward water electrolysis that can work at high current density (≥1000 mA cm-2) are urgently needed. Herein, Mn-doped RuO2 (MnxRu1-xO2) nanofibers (NFs) are constructed to achieve this object, presenting wonderful hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) performances with the overpotentials of only 269 and 461 mV at 1 A cm-2 in 1 m KOH solution, and remarkably stability under industrial demand with 1 A cm-2, significantly better than the benchmark Pt/C and commercial RuO2 electrocatalysts, respectively. More importantly, the assembled Mn0.05Ru0.95O2 NFs||Mn0.05Ru0.95O2 NFs electrolyzer toward overall water splitting reaches the current density of 10 mA cm-2 with a cell voltage of 1.52 V and also delivers an outstanding stability over 150 h of continuous operation, far surpassing commercial Pt/C||commercial RuO2, RuO2 NFs||RuO2 NFs and most previously reported exceptional electrolyzers. Theoretical calculations indicate that Mn-doping into RuO2 can significantly optimize the electronic structure and weaken the strength of O─H bond to achieve the near-zero hydrogen adsorption free energy (ΔGH*) value for HER, and can also effectively weaken the adsorption strength of intermediate O* at the relevant sites, achieving the higher OER catalytic activity, since the overlapping center of p-d orbitals is closer to the Fermi level.

CFAGO: cross-fusion of network and attributes based on attention mechanism for protein function prediction.

MOTIVATION: Protein function annotation is fundamental to understanding biological mechanisms. The abundant genome-scale protein-protein interaction (PPI) networks, together with other protein biological attributes, provide rich information for annotating protein functions. As PPI networks and biological attributes describe protein functions from different perspectives, it is highly challenging to cross-fuse them for protein function prediction. Recently, several methods combine the PPI networks and protein attributes via the graph neural networks (GNNs). However, GNNs may inherit or even magnify the bias caused by noisy edges in PPI networks. Besides, GNNs with stacking of many layers may cause the over-smoothing problem of node representations. RESULTS: We develop a novel protein function prediction method, CFAGO, to integrate single-species PPI networks and protein biological attributes via a multi-head attention mechanism. CFAGO is first pre-trained with an encoder-decoder architecture to capture the universal protein representation of the two sources. It is then fine-tuned to learn more effective protein representations for protein function prediction. Benchmark experiments on human and mouse datasets show CFAGO outperforms state-of-the-art single-species network-based methods by at least 7.59%, 6.90%, 11.68% in terms of m-AUPR, M-AUPR, and Fmax, respectively, demonstrating cross-fusion by multi-head attention mechanism can greatly improve the protein function prediction. We further evaluate the quality of captured protein representations in terms of Davies Bouldin Score, whose results show that cross-fused protein representations by multi-head attention mechanism are at least 2.7% better than that of original and concatenated representations. We believe CFAGO is an effective tool for protein function prediction. AVAILABILITY AND IMPLEMENTATION: The source code of CFAGO and experiments data are available at: http://bliulab.net/CFAGO/.

mir-605-3p prevents liver premetastatic niche formation by inhibiting angiogenesis via decreasing exosomal nos3 release in gastric cancer.

BACKGROUND: Cancer-induced pre-metastatic niches (PMNs) play a decisive role in promoting metastasis by facilitating angiogenesis in distant sites. Evidence accumulates suggesting that microRNAs (miRNAs) exert significant influence on angiogenesis during PMN formation, yet their specific roles and regulatory mechanisms in gastric cancer (GC) remain underexplored. METHODS: miR-605-3p was identified through miRNA-seq and validated by qRT-PCR. Its correlation with the clinicopathological characteristics and prognosis was analyzed in GC. Functional assays were performed to examine angiogenesis both in vitro and in vivo. The related molecular mechanisms were elucidated using RNA-seq, immunofluorescence, transmission electron microscopy, nanoparticle tracking analysis, enzyme-linked immunosorbent assay, luciferase reporter assays and bioinformatics analysis. RESULTS: miR-605-3p was screened as a candidate miRNA that may regulate angiogenesis in GC. Low expression of miR-605-3p is associated with shorter overall survival and disease-free survival in GC. miR-605-3p-mediated GC-secreted exosomes regulate angiogenesis by regulating exosomal nitric oxide synthase 3 (NOS3) derived from GC cells. Mechanistically, miR-605-3p reduced the secretion of exosomes by inhibiting vesicle-associated membrane protein 3 (VAMP3) expression and affects the transport of multivesicular bodies to the GC cell membrane. At the same time, miR-605-3p reduces NOS3 levels in exosomes by inhibiting the expression of intracellular NOS3. Upon uptake of GC cell-derived exosomal NOS3, human umbilical vein endothelial cells exhibited increased nitric oxide levels, which induced angiogenesis, established liver PMN and ultimately promoted the occurrence of liver metastasis. Furthermore, a high level of plasma exosomal NOS3 was clinically associated with metastasis in GC patients. CONCLUSIONS: miR-605-3p may play a pivotal role in regulating VAMP3-mediated secretion of exosomal NOS3, thereby affecting the formation of GC PMN and thus inhibiting GC metastasis.

Integrative transcriptomics and cell systems analyses reveal protective pathways controlled by Igfbp-3 in anthracycline-induced cardiotoxicity.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapeutic agents; however, their use is hampered by subsequent cardiotoxicity risk. Our understanding of cardiomyocyte protective pathways activated following anthracycline-induced cardiotoxicity (AIC) remains incomplete. Insulin-like growth factor binding protein (IGFBP) 3 (Igfbp-3), the most abundant IGFBP family member in the circulation, is associated with effects on the metabolism, proliferation, and survival of various cells. Whereas Igfbp-3 is induced by Dox in the heart, its role in AIC is ill-defined. We investigated molecular mechanisms as well as systems-level transcriptomic consequences of manipulating Igfbp-3 in AIC using neonatal rat ventricular myocytes and human-induced pluripotent stem cell-derived cardiomyocytes. Our findings reveal that Dox induces the nuclear enrichment of Igfbp-3 in cardiomyocytes. Furthermore, Igfbp-3 reduces DNA damage, impedes topoisomerase IIß expression (Top2ß) which forms Top2ß-Dox-DNA cleavage complex leading to DNA double-strand breaks (DSB), alleviates detyrosinated microtubule accumulation-a hallmark of increased cardiomyocyte stiffness and heart failure-and favorably affects contractility following Dox treatment. These results indicate that Igfbp-3 is induced by cardiomyocytes in an effort to mitigate AIC.

Mutations of epigenetic modifier genes predict poor outcome in adult acute lymphoblastic leukemia.

Epigenetic modifier (EM) genes play important roles in the occurrence and progression of acute lymphoblastic leukemia (ALL). However, the prognostic significance of EM mutations in ALL has not yet been thoroughly investigated. This retrospective study included 205 adult patients with ALL engaged in a pediatric-type regimen. Based on targeted next-generation sequencing, they were divided into EM mutation group (EM-mut, n = 75) and EM wild-type group (EM-wt, n = 130). The EM-mut group showed a higher positive rate of minimal residual disease (MRD) on treatment day24 and before consolidation therapy (P = 0.026, 0.020). Multivariate Cox regression analysis showed that EM-mut was an independent adverse factor for overall survival (OS) and event-free survival (EFS) (HR = 2.123, 1.742; P = 0.009, 0.007). Survival analysis revealed that the OS and EFS rates were significantly lower in the EM-mut group than in the EM-wt group (3-year OS rate, 45.8% vs. 65.0%, P = 0.0041; 3-year EFS rate, 36.7% vs. 53.2%, P = 0.011). In conclusion, EM was frequently mutated in adult ALL and was characterized by poor response to induction therapy and inferior clinical outcomes.

Highly Efficient Transformation of Tar Model Compounds into Hydrogen by a Ni-Co Alloy Nanocatalyst During Tar Steam Reforming.

Hydrogen (H2) production from coal and biomass gasification was considered a long-term and viable way to solve energy crises and global warming. Tar, generated as a hazardous byproduct, limited its large-scale applications by clogging and corroding gasification equipment. Although catalytic steam reforming technology was used to convert tar into H2, catalyst deactivation restricted its applicability. A novel nanocatalyst was first synthesized by the modified sol-gel method using activated biochar as the support, nickel (Ni) as the active component, and cobalt (Co) as the promoter for converting tar into H2. The results indicated that a high H2 yield of 263.84 g H2/kg TMCs (Tar Model Compounds) and TMC conversion of almost 100% were obtained over 6% Ni-4% Co/char, with more than 30% increase in hydrogen yield compared to traditional catalysts. Moreover, 6% Ni-4% Co/char exhibited excellent resistance to carbon deposition by removing the nucleation sites for graphite formation, forming stable Ni-Co alloy, and promoting the char gasification reaction; resistance to oxidation deactivation due to the high oxygen affinity of Co and reduction of the oxidized nickel by H2 and CO; resistance to sintering deactivation by strengthened interaction between Ni and Co, high specific surface area (920.61 m2/g), and high dispersion (7.3%) of Ni nanoparticles. This work provided a novel nanocatalyst with significant potential for long-term practical applications in the in situ conversion of tar into H2 during steam reforming.

Double pyramid stacked CoO nano-crystals induced by graphene at low temperatures as highly efficient Fenton-like catalysts.

Transition metal oxides are widely used as Fenton-like catalysts in the treatment of organic pollutants, but their synthesis usually requires a high temperature. Herein, an all-solid-state synthesis method controlled by graphene was used to prepare a double pyramid stacked CoO nano-crystal at a low temperature. The preparation temperature decreased by 200 °C (over 30% reduction) due to the introduction of graphene, largely reducing the reaction energy barrier. Interestingly, the corresponding degradation rate constants (kobs) of this graphene-supported pyramid CoO nano-crystals for organic molecules after their adsorption were over 2.5 and 35 times higher than that before adsorption and that of free CoO, respectively. This high catalytic efficiency is attributed to the adsorption of pollutants at the surface by supporting graphene layers, while free radicals activated by CoO can directly and rapidly contact and degrade them. These findings provide a new strategy to prepare low carbon-consuming transition metal oxides for highly efficient Fenton-like catalysts.