Mammalian SIRT6 Represses Invasive Cancer Cell Phenotypes through ATP Citrate Lyase (ACLY)-Dependent Histone Acetylation.

The modulation of dynamic histone acetylation states is key for organizing chromatin structure and modulating gene expression and is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes. The mammalian SIRT6 protein, a member of the Class III HDAC Sirtuin family of NAD+-dependent enzymes, plays pivotal roles in aging, metabolism, and cancer biology. Through its site-specific histone deacetylation activity, SIRT6 promotes chromatin silencing and transcriptional regulation of aging-associated, metabolic, and tumor suppressive gene expression programs. ATP citrate lyase (ACLY) is a nucleo-cytoplasmic enzyme that produces acetyl coenzyme A (acetyl-CoA), which is the required acetyl donor for lysine acetylation by HATs. In addition to playing a central role in generating cytosolic acetyl-CoA for de novo lipogenesis, a growing body of work indicates that ACLY also functions in the nucleus where it contributes to the nutrient-sensitive regulation of nuclear acetyl-CoA availability for histone acetylation in cancer cells. In this study, we have identified a novel function of SIRT6 in controlling nuclear levels of ACLY and ACLY-dependent tumor suppressive gene regulation. The inactivation of SIRT6 in cancer cells leads to the accumulation of nuclear ACLY protein and increases nuclear acetyl-CoA pools, which in turn drive locus-specific histone acetylation and the expression of cancer cell adhesion and migration genes that promote tumor invasiveness. Our findings uncover a novel mechanism of SIRT6 in suppressing invasive cancer cell phenotypes and identify acetyl-CoA responsive cell migration and adhesion genes as downstream targets of SIRT6.

Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient ß-catenin degradation.

The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scaffolds GSK3ß and CK1 to earmark ß-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of ß-catenin and oncogenesis. However, the molecular mechanism by which APC promotes ß-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple ß-catenin and Axin interacting sites, undergoes liquid-liquid phase separation (LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and ß-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the ß-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of ß-catenin.

Methyltransferase-like 21C (METTL21C) methylates alanine tRNA synthetase at Lys-943 in muscle tissue.

Protein-lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70-kDa protein 8 (HSPA8). However, a clear in vitro methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate(s) in vivo is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine tRNA synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both in vitro and in vivoIn vitro METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells depends upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a bona fide in vitro substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.

SETD5-Coordinated Chromatin Reprogramming Regulates Adaptive Resistance to Targeted Pancreatic Cancer Therapy.

Molecular mechanisms underlying adaptive targeted therapy resistance in pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Here, we identify SETD5 as a major driver of PDAC resistance to MEK1/2 inhibition (MEKi). SETD5 is induced by MEKi resistance and its deletion restores refractory PDAC vulnerability to MEKi therapy in mouse models and patient-derived xenografts. SETD5 lacks histone methyltransferase activity but scaffolds a co-repressor complex, including HDAC3 and G9a. Gene silencing by the SETD5 complex regulates known drug resistance pathways to reprogram cellular responses to MEKi. Pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustains PDAC tumor growth inhibition in vivo. Our work uncovers SETD5 as a key mediator of acquired MEKi therapy resistance in PDAC and suggests a context for advancing MEKi use in the clinic.

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules.

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein ß-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

The epigenetic regulator SIRT7 guards against mammalian cellular senescence induced by ribosomal DNA instability.

In the yeast Saccharomyces cerevisiae, genomic instability in rDNA repeat sequences is an underlying cause of cell aging and is suppressed by the chromatin-silencing factor Sir2. In humans, rDNA instability is observed in cancers and premature aging syndromes, but its underlying mechanisms and functional consequences remain unclear. Here, we uncovered a pivotal role of sirtuin 7 (SIRT7), a mammalian Sir2 homolog, in guarding against rDNA instability and show that this function of SIRT7 protects against senescence in primary human cells. We found that, mechanistically, SIRT7 is required for association of SNF2H (also called SMARCA5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A, member 5), a component of the nucleolar heterochromatin-silencing complex NoRC, with rDNA sequences. Defective rDNA-heterochromatin silencing in SIRT7-deficient cells unleashed rDNA instability, with excision and loss of rDNA gene copies, which in turn induced acute senescence. Mounting evidence indicates that accumulation of senescent cells significantly contributes to tissue dysfunction in aging-related pathologies. Our findings identify rDNA instability as a driver of mammalian cellular senescence and implicate SIRT7-dependent heterochromatin silencing in protecting against this process.

Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules.

Sirtuins are protein deacylases regulating metabolism and stress responses, and are implicated in aging-related diseases. Small molecule activators for the human sirtuins Sirt1-7 are sought as chemical tools and potential therapeutics, such as for cancer. Activators are available for Sirt1 and exploit its unique N-terminus, whereas drug-like activators for Sirt2-7 are lacking. We synthesized and screened pyrrolo[1,2-a]quinoxaline derivatives, yielding the first synthetic Sirt6 activators. Biochemical assays show direct, substrate-independent compound binding to the Sirt6 catalytic core and potent activation of Sirt6-dependent deacetylation of peptide substrates and complete nucleosomes. Crystal structures of Sirt6/activator complexes reveal that the compounds bind to a Sirt6-specific acyl channel pocket and identify key interactions. Our results establish potent Sirt6 activation with small molecules and provide a structural basis for further development of Sirt6 activators as tools and therapeutics.

Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins.

Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration.

No Significant Increase in the Δ4- and Δ7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation.

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants.

E2F transcription factor 1 regulates cellular and organismal senescence by inhibiting Forkhead box O transcription factors.

E2F1 and FOXO3 are two transcription factors that have been shown to participate in cellular senescence. Previous report reveals that E2F1 enhanced cellular senescence in human fibroblast cells, while FOXO transcription factors play against senescence by regulation reactive oxygen species scavenging proteins. However, their functional interplay has been unclear. Here we use E2F1 knock-out murine Embryonic fibroblasts (MEFs), knockdown RNAi constructs, and ectopic expression of E2F1 to show that it functions by negatively regulating FOXO3. E2F1 attenuates FOXO3-mediated expression of MnSOD and Catalase without affecting FOXO3 protein stability, subcellular localization, or phosphorylation by Akt. We mapped the interaction between E2F1 and FOXO3 to a region including the DNA binding domain of E2F1 and the C-terminal transcription-activation domain of FOXO3. We propose that E2F1 inhibits FOXO3-dependent transcription by directly binding FOXO3 in the nucleus and preventing activation of its target genes. Moreover, knockdown of the Caenorhabditis elegans E2F1 ortholog efl-1 significantly extends lifespan in a manner that requires the activity of the C. elegans FOXO gene daf-16. We conclude that there is an evolutionarily conserved signaling connection between E2F1 and FOXO3, which regulates cellular senescence and aging by regulating the activity of FOXO3. We speculate that drugs and/or therapies that inhibit this physical interaction might be good candidates for reducing cellular senescence and increasing longevity.

Absolute quantification of a steroid hormone that regulates development in Caenorhabditis elegans.

Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-ß, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.

Characterization of PUD-1 and PUD-2, two proteins up-regulated in a long-lived daf-2 mutant.

C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar ß-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid ß-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against Aß.

Controllable preferential-etching synthesis of ZnO nanotube arrays on SiO2 substrate for solid-phase microextraction.

An improved route to obtain ZnO nanotube arrays and its first application to headspace solid-phase microextraction (HSSPME) as an adsorptive coating were described. The ZnO nanotube arrays were synthesized by a two-step chemical process including the hydrothermal synthesis of ZnO nanorod arrays on the surface of silica fiber (SiO(2)) in the first step, and the formation of ZnO nanotubes by selectively etching in NH(3)·H(2)O solution in the second step. The influence of NH(3)·H(2)O concentration, etching time, reaction temperature, and aging time in the ZnO nanotubes formation process was investigated, and arrays of ZnO nanotube with tailored dimensions (250 nm external diameters, 70 nm wall thicknesses and 2 µm lengths) could be obtained by varying the conditions. In addition, the feasibility of ZnO nanotube arrays adopted for HSSPME was evaluated by extracting volatile organic compounds (VOCs) by use of benzene, toluene, ethylbenzene, o-, m-and p-xylene (BTEX) as model compounds and the results showed that the coating has good extraction capability. The analytes were linear in the range of 10-600 µg L(-1) (r > 0.9960) and the detection limits were about 0.005-0.01 µg L(-1), lower than that obtained with ZnO nanorod arrays. The relative standard derivations (RSD) for the repeatability of single fiber and fiber-to-fiber were lower than 9.5% and 13.8%, respectively. The prepared coating showed good recoveries in the range of 87%-108% and long lifetime (more than 50 times), implying to be a potential absorbent for the VOCs in water samples.