Inhibition of EHMT1/2 rescues synaptic damage and motor impairment in a PD mouse model.

Epigenetic dysregulation that leads to alterations in gene expression and is suggested to be one of the key pathophysiological factors of Parkinson's disease (PD). Here, we found that α-synuclein preformed fibrils (PFFs) induced histone H3 dimethylation at lysine 9 (H3K9me2) and increased the euchromatic histone methyltransferases EHMT1 and EHMT2, which were accompanied by neuronal synaptic damage, including loss of synapses and diminished expression levels of synaptic-related proteins. Furthermore, the levels of H3K9me2 at promoters in genes that encode the synaptic-related proteins SNAP25, PSD95, Synapsin 1 and vGLUT1 were increased in primary neurons after PFF treatment, which suggests a linkage between H3K9 dimethylation and synaptic dysfunction. Inhibition of EHMT1/2 with the specific inhibitor A-366 or shRNA suppressed histone methylation and alleviated synaptic damage in primary neurons that were treated with PFFs. In addition, the synaptic damage and motor impairment in mice that were injected with PFFs were repressed by treatment with the EHMT1/2 inhibitor A-366. Thus, our findings reveal the role of histone H3 modification by EHMT1/2 in synaptic damage and motor impairment in a PFF animal model, suggesting the involvement of epigenetic dysregulation in PD pathogenesis.

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis.

Shugoshin-1 (Sgo1) is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis. It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120. However, it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis. Here, we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis. Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A. Interestingly, the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere, resulting in aberrant chromosome segregation and premature chromatid separation. Taken together, these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1-CENP-A interaction guides accurate cell division.

Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis.

In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.

Systematic characterization of chromodomain proteins reveals an H3K9me1/2 reader regulating aging in C. elegans.

The chromatin organization modifier domain (chromodomain) is an evolutionally conserved motif across eukaryotic species. The chromodomain mainly functions as a histone methyl-lysine reader to modulate gene expression, chromatin spatial conformation and genome stability. Mutations or aberrant expression of chromodomain proteins can result in cancer and other human diseases. Here, we systematically tag chromodomain proteins with green fluorescent protein (GFP) using CRISPR/Cas9 technology in C. elegans. By combining ChIP-seq analysis and imaging, we delineate a comprehensive expression and functional map of chromodomain proteins. We then conduct a candidate-based RNAi screening and identify factors that regulate the expression and subcellular localization of the chromodomain proteins. Specifically, we reveal an H3K9me1/2 reader, CEC-5, both by in vitro biochemistry and in vivo ChIP assays. MET-2, an H3K9me1/2 writer, is required for CEC-5 association with heterochromatin. Both MET-2 and CEC-5 are required for the normal lifespan of C. elegans. Furthermore, a forward genetic screening identifies a conserved Arginine124 of CEC-5's chromodomain, which is essential for CEC-5's association with chromatin and life span regulation. Thus, our work will serve as a reference to explore chromodomain functions and regulation in C. elegans and allow potential applications in aging-related human diseases.

Enhanced imaging of endogenous metabolites by negative ammonia assisted DESI/PI mass spectrometry.

In this work, endogenous metabolites in mouse brain tissue were imaged by negative desorption electrospray ionization (DESI), ammonia assisted DESI (aa-DESI), DESI/post-photoionization (DESI/PI), and ammonia assisted DESI/PI (aa-DESI/PI) mass spectrometry imaging (MSI) strategies. The combined effect of ammonia additive and post-photoionization was found to play an important role in the enhancement of sensitivity and coverage for endogenous analytes under ambient conditions. Compared with DESI, aa-DESI/PI can provide increased signal intensities for metabolites up to 37.1-fold, as well as the imaging of nine more small metabolites (m/z < 350) (26 for aa-DESI/PI and 17 for DESI) in mouse brain tissue. The results of Pearson correlation analysis and KEGG pathway analysis showed that the enhanced imaging strategy of aa-DESI/PI can facilitate the study of endogenous metabolic pathways and molecular networks. Moreover, the imaging results of mouse tumor tissue demonstrated the promising application of aa-DESI/PI in tumor research.

Phase separation of EB1 guides microtubule plus-end dynamics.

In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid-liquid phase separation (LLPS) to constitute the microtubule plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a compartment for enriching tubulin dimers and other plus-end tracking proteins. Real-time imaging of chromosome segregation in HeLa cells expressing LLPS-deficient EB1 mutants revealed the importance of EB1 LLPS dynamics in mitotic chromosome movements. These findings demonstrate that EB1 forms a distinct physical and biochemical membraneless-organelle via multivalent interactions that guide microtubule dynamics.

Structural insights into human CCAN complex assembled onto DNA.

In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

Staphylococcal superantigen-like protein 10 induces necroptosis through TNFR1 activation of RIPK3-dependent signal pathways.

Staphylococcal aureus (S. aureus) infection can lead to a wide range of diseases such as sepsis and pneumonia. Staphylococcal superantigen-like (SSL) proteins, expressed by all known S. aureus strains, are shown to be involved in immune evasion during S. aureus infection. Here, we show that SSL10, an SSL family protein, exhibits potent cytotoxicity against human cells (HEK293T and HUVEC) by inducing necroptosis upon binding to its receptor TNFR1 on the cell membrane. After binding, two distinct signaling pathways are activated downstream of TNFR1 in a RIPK3-dependent manner, i.e., the RIPK1-RIPK3-MLKL and RIPK3-CaMKII-mitochondrial permeability transition pore (mPTP) pathways. Knockout of ssl10 in S. aureus significantly reduces cytotoxicity of the culture supernatants of S. aureus, indicating that SSL10 is involved in extracellular cytotoxicity during infection. We determined the crystal structure of SSL10 at 1.9 Å resolution and identified a positively charged surface of SSL10 responsible for TNFR1 binding and cytotoxic activity. This study thus provides the description of cytotoxicity through induction of necroptosis by the SSL10 protein, and a potential target for clinical treatment of S. aureus-associated diseases.

GAF domain is essential for nitrate-dependent AtNLP7 function.

Nitrate is an essential nutrient and an important signaling molecule in plants. However, the molecular mechanisms by which plants perceive nitrate deficiency signaling are still not well understood. Here we report that AtNLP7 protein transport from the nucleus to the cytoplasm in response to nitrate deficiency is dependent on the N-terminal GAF domain. With the deletion of the GAF domain, AtNLP7ΔGAF always remains in the nucleus regardless of nitrate availability. AtNLP7 ΔGAF also shows reduced activation of nitrate-induced genes due to its impaired binding to the nitrate-responsive cis-element (NRE) as well as decreased growth like nlp7-1 mutant. In addition, AtNLP7ΔGAF is unable to mediate the reduction of reactive oxygen species (ROS) accumulation upon nitrate treatment. Our investigation shows that the GAF domain of AtNLP7 plays a critical role in the sensing of nitrate deficiency signal and in the nitrate-triggered ROS signaling process.

Baicalein Acts against Candida albicans by Targeting Eno1 and Inhibiting Glycolysis.

Baicalein (BE) is a promising antifungal small-molecule compound with an extended antifungal spectrum, good synergy with fluconazole, and low toxicity, but its target protein and antifungal mechanism remain elusive. In this study, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. Eno1 acts as a key therapeutic target of the drug, as BE had no antifungal activity against the eno1 null mutant in a Galleria mellonella model of C. albicans infection. To investigate the mechanism of action, we solved the crystal structure of C. albicans Eno1(CaEno1) and then compared the difference between this structure and that of Eno1 from humans. The predicted primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Both positions S269 and K273 have different residues in the human Eno1 (hEno1). This finding suggests that BE may bind selectively to CaEno1, which would limit the potential for side effects in humans. Our findings demonstrate that Eno1 is a target protein of BE and thus may serve as a novel target for the development of antifungal therapeutics acting through the inhibition of glycolysis. IMPORTANCE Baicalein (BE) is a promising antifungal agent which has been well characterized, but its target protein is still undiscovered. The protein Eno1 plays a crucial role in the survival of Candida albicans. However, there are few antifungal agents which inhibit the functions of Eno1. Here, we found that BE can function against Candida albicans by disrupting glycolysis through targeting Eno1 and inhibiting its function. We further solved the crystal structure of C. albicans Eno1(CaEno1) and predicted that the primary binding site of BE on CaEno1 is between amino acids D261 and W274, with D263, S269, and K273 playing critical roles in the interaction with BE. Our findings will be helpful to get specific small-molecule inhibitors of CaEno1 and open the way for the development of new antifungal therapeutics targeted at inhibiting glycolysis.

Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis.

Spindle position control is essential for cell fate determination and organogenesis. Early studies indicate the essential role of the evolutionarily conserved Gαi/LGN/NuMA network in spindle positioning. However, the regulatory mechanisms that couple astral microtubules dynamics to the spindle orientation remain elusive. Here we delineated a new mitosis-specific crotonylation-regulated astral microtubule-EB1-NuMA interaction in mitosis. EB1 is a substrate of TIP60, and TIP60-dependent crotonylation of EB1 tunes accurate spindle positioning in mitosis. Mechanistically, TIP60 crotonylation of EB1 at Lys66 forms a dynamic link between accurate attachment of astral microtubules to the lateral cell cortex defined by NuMA-LGN and fine tune of spindle positioning. Real-time imaging of chromosome movements in HeLa cells expressing genetically encoded crotonylated EB1 revealed the importance of crotonylation dynamics for accurate control of spindle orientation during metaphase-anaphase transition. These findings delineate a general signaling cascade that integrates protein crotonylation with accurate spindle positioning for chromosome stability in mitosis.

Resveratrol-induced Sirt1 phosphorylation by LKB1 mediates mitochondrial metabolism.

The NAD+-dependent deacetylase Sirt1 has been implicated in the prevention of many age-related diseases, including cancer, type 2 diabetes, and cardiovascular disease. Resveratrol, a plant polyphenol, exhibits antiaging, antitumor, and vascular protection effects by activating Sirt1. However, the molecular mechanism of Sirt1 activation as induced by resveratrol remains unclear. By knockdown/rescue experiments, fluorometric Sirt1 activity assay, immunoprecipitation, and pull-down assays, we identify here that the tumor suppressor LKB1 (liver kinase B1) as a direct activator of Sirt1 elicited by resveratrol. Resveratrol promotes the binding between LKB1 and Sirt1, which we first reported, and this binding leads to LKB1-mediated phosphorylation of Sirt1 at three different serine residues in the C terminus of Sirt1. Mechanistically, LKB1-mediated phosphorylation increases intramolecular interactions in Sirt1, such as the binding of the C terminus to the deacetylase core domain, thereby eliminating DBC1 (Deleted in Breast Cancer 1, Sirt1 endogenous inhibitor) inhibition and promoting Sirt1-substrate interaction. Functionally, LKB1-dependent Sirt1 activation increases mitochondrial biogenesis and respiration through deacetylation and activation of the transcriptional coactivator PGC-1α. These results identify Sirt1 as a context-dependent target of LKB1 and suggest that a resveratrol-stimulated LKB1-Sirt1 pathway plays a vital role in mitochondrial metabolism, a key physiological process that contributes to numerous age-related diseases.

Structural insights into the recognition of histone H3Q5 serotonylation by WDR5.

Serotonylation of histone H3Q5 (H3Q5ser) is a recently identified posttranslational modification of histones that acts as a permissive marker for gene activation in synergy with H3K4me3 during neuronal cell differentiation. However, any proteins that specifically recognize H3Q5ser remain unknown. Here, we found that WDR5 interacts with the N-terminal tail of histone H3 and functions as a "reader" for H3Q5ser. Crystal structures of WDR5 in complex with H3Q5ser and H3K4me3Q5ser peptides revealed that the serotonyl group is accommodated in a shallow surface pocket of WDR5. Experiments in neuroblastoma cells demonstrate that H3K4me3 modification is hampered upon disruption of WDR5-H3Q5ser interaction. WDR5 colocalizes with H3Q5ser in the promoter regions of cancer-promoting genes in neuroblastoma cells, where it promotes gene transcription to induce cell proliferation. Thus, beyond revealing a previously unknown mechanism through which WDR5 reads H3Q5ser to activate transcription, our study suggests that this WDR5-H3Q5ser-mediated epigenetic regulation apparently promotes tumorigenesis.

Crystal structures of NAD+-linked isocitrate dehydrogenase from the green alga Ostreococcus tauri and its evolutionary relationship with eukaryotic NADP+-linked homologs.

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.

AMPKα2 activation by an energy-independent signal ensures chromosomal stability during mitosis.

AMP-activated protein kinase (AMPK) senses energy status and impacts energy-consuming events by initiating metabolism regulatory signals in cells. Accumulating evidences suggest a role of AMPK in mitosis regulation, but the mechanism of mitotic AMPK activation and function remains elusive. Here we report that AMPKα2, but not AMPKα1, is sequentially phosphorylated and activated by CDK1 and PLK1, which enables AMPKα2 to accurately guide chromosome segregation in mitosis. Phosphorylation at Thr485 by activated CDK1-Cyclin B1 brings the ST-stretch of AMPKα2 to the Polo box domain of PLK1 for subsequent Thr172 phosphorylation by PLK1. Inserting of the AMPKα2 ST-stretch into AMPKα1, which lacks the ST-stretch, can correct mitotic chromosome segregation defects in AMPKα2-depleted cells. These findings uncovered a specific signaling cascade integrating sequential phosphorylation by CDK1 and PLK1 of AMPKα2 with mitosis to maintain genomic stability, thus defining an isoform-specific AMPKα2 function, which will facilitate future research on energy sensing in mitosis.

Structural insights into the intermolecular interaction of the adhesin SdrC in the pathogenicity of Staphylococcus aureus.

Staphylococcus aureus is an opportunistic disease-causing pathogen that is widely found in the community and on medical equipment. A series of virulence factors secreted by S. aureus can trigger severe diseases such as sepsis, endocarditis and toxic shock, and thus have a great impact on human health. The transformation of S. aureus from a colonization state to a pathogenic state during its life cycle is intimately associated with the initiation of bacterial aggregation and biofilm accumulation. SdrC, an S. aureus surface protein, can act as an adhesin to promote cell attachment and aggregation by an unknown mechanism. Here, structural studies demonstrate that SdrC forms a unique dimer through intermolecular interaction. It is proposed that the dimerization of SdrC enhances the efficiency of bacteria-host attachment and therefore contributes to the pathogenicity of S. aureus.

Centromere targeting of Mis18 requires the interaction with DNA and H2A-H2B in fission yeast.

Faithful chromosome segregation during mitosis requires the correct assembly of kinetochore on the centromere. CENP-A is a variant of histone H3, which specializes the centromere region on chromatin and mediates the kinetochore assembly. The Mis18 complex plays a critical role in initiating the centromere loading of the newly-synthesized CENP-A. However, it remains unclear how Mis18 complex (spMis18, spMis16 and spMis19) is located to the centromere to license the recruitment of Cnp1CENP-A in Schizosaccharomyces pombe. We found that spMis18 directly binds to nucleosomal DNA through its extreme C-terminus and interacts with H2A-H2B dimer via the acidic region on the surface of its Yippee-like domain. Live-cell imaging confirmed that mutation of the acidic region and deletion of the extreme C-terminus significantly impairs the localization of spMis18 and Cnp1 to the centromere and delays chromosome segregation during mitosis. Our findings illustrate that the interaction of spMis18 with histone H2A-H2B and DNA plays important roles in the recruitment of spMis18 and Cnp1 to the centromere in fission yeast.

WDR63 inhibits Arp2/3-dependent actin polymerization and mediates the function of p53 in suppressing metastasis.

Accumulating evidence suggests that p53 plays a suppressive role in cancer metastasis, yet the underlying mechanism remains largely unclear. Regulation of actin dynamics is essential for the control of cell migration, which is an important step in metastasis. The Arp2/3 complex is a major nucleation factor to initiate branched actin polymerization that drives cell migration. However, it is unknown whether p53 could suppress metastasis through modulating Arp2/3 function. Here, we report that WDR63 is transcriptionally upregulated by p53. We show with migration assays and mouse xenograft models that WDR63 negatively regulates cell migration, invasion, and metastasis downstream of p53. Mechanistically, WDR63 interacts with the Arp2/3 complex and inhibits Arp2/3-mediated actin polymerization. Furthermore, WDR63 overexpression is sufficient to dampen the increase in cell migration, invasion, and metastasis induced by p53 depletion. Together, these findings suggest that WDR63 is an important player in the regulation of Arp2/3 function and also implicate WDR63 as a critical mediator of p53 in suppressing metastasis.