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The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila
Kenneth Kin Lam Wong, Ta-Wei Liu, Jessica M. Parker, Donald A. R. Sinclair, Yi-Yun Chen, Kay-Hooi Khoo, View ORCID ProfileDavid J. Vocadlo, and View ORCID ProfileEsther M. Verheyen
PNAS first published January 13, 2020 https://doi.org/10.1073/pnas.1912894117
Kenneth Kin Lam Wong
aDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;bCentre for Cell Biology, Development and Disease, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;
Ta-Wei Liu
cDepartment of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;
Jessica M. Parker
aDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;
Donald A. R. Sinclair
Yi-Yun Chen
dInstitute of Biological Chemistry, Academia Sinica, 115 Taipei, Taiwan
Kay-Hooi Khoo
David J. Vocadlo
aDepartment of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;bCentre for Cell Biology, Development and Disease, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;cDepartment of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;
ORCID record for David J. Vocadlo
Esther M. Verheyen
ORCID record for Esther M. Verheyen
For correspondence: everheye@sfu.ca
Edited by John A. Hanover, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, and accepted by Editorial Board Member Kathryn V. Anderson December 9, 2019 (received for review July 29, 2019)
O-GlcNAc transferase (OGT) transfers the sugar O-GlcNAc to serine and threonine residues of target proteins, a process known as O-GlcNAcylation. Using Drosophila as an animal model, we identify the protein kinase Hipk as a target protein for OGT. Further biochemical assays and genetic interaction studies reveal that OGT plays critical roles in controlling Hipk protein stability and Hipk-induced tissue growth abnormalities. This regulation is likely operative in mammals as we find that human HIPK2 is less stable when it cannot be O-GlcNAc modified. HIPK2 is implicated in several cancers and chronic fibrosis. Our work therefore opens up the possibility of exploiting the regulatory role of OGT in the treatment of HIPK2-related disorders.
Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.
OGT
O-GlcNAc
Nutrients such as glucose, amino acids, and fatty acids are metabolic fuels that provide energy to cells (1). They also function as signaling molecules in various nutrient signaling pathways (1), allowing coordination between nutrient sensing and cellular behaviors like cell growth (2). One emerging nutrient signaling pathway is the hexosamine biosynthetic pathway (HBP) (3). In the HBP, glutamine:fructose-6-phosphate amidotransferases (GFATs) catalyze the first and rate-limiting step that converts glucose-derived fructose-6-phosphate into glucosamine-6-phosphate (3) (Fig. 1A). The end product of the HBP is UDP-GlcNAc, the donor nucleotide sugar substrate for various glycosyltransferases, including nucleocytoplasmic O-GlcNAc transferase (OGT) (4). OGT is the only known enzyme that catalyzes the addition of O-GlcNAc to serine and threonine residues of hundreds of target proteins in a UDP-GlcNAc–dependent manner (5). Notably, O-GlcNAcase (OGA) removes O-GlcNAc from proteins, making O-GlcNAc a dynamic and reversible posttranslational modification (PTM) (6). In this way, cellular UDP-GlcNAc levels and global O-GlcNAcylation are coordinated and are highly responsive to glucose availability, making O-GlcNAc well suited to serve as a nutrient-sensing mechanism (7, 8).
High dietary sugar potentiates Hipk-induced growth abnormalities through HBP and OGT. (A) Schematic diagram of the HBP. Dietary glucose and glucosamine are shown in blue, metabolites in gray, enzymes in red. (B–D) Adult legs of hipk-overexpressing animals (dpp > hipk) which were fed a control diet (0.15 M sucrose) (B), a HSD (1 M sucrose) (C), or a glucosamine-supplemented diet (D) from embryo hatch. White arrowheads mark the WT-like, slender leg phenotype. Black arrowheads mark the mild leg malformation phenotype. (E) A violin plot showing the malformed leg phenotype of the dpp > hipk flies raised on different diets. The letters B–D refer to the flies shown in Fig. 1 B–D. Numbers of flies counted N: (B) 173, (C) 52, and (D) 37. P values were calculated using unpaired two-tailed Student's t test. (F–I) Adult legs of dpp > hipk + GFP (F), dpp > hipk + gfat1-RNAi + gfat2-RNAi (G), dpp > hipk + ogt-RNAi (H), and dpp > hipk + ogt-RNAi #2 (I) flies which were fed an HSD from embryo hatch. Black arrowheads mark the disfigured legs. White arrowheads mark the WT-like legs. (J) A violin plot showing the malformed leg phenotype of the indicated animals fed a HSD. The letters F–I refer to the flies shown in Fig. 1 F–I. Numbers of flies counted N: (F) 53, (G) 28, (H) 25, and (I) 29. P values were calculated using unpaired two-tailed Student's t test. Flies were raised at 25 °C with the indicated diets. (Scale bars, 500 μm.)
Pathologically, O-GlcNAc has been implicated in cancers. For instance, hyper–O-GlcNAcylation, high levels of OGT or GFAT2, or low OGA levels are positively correlated with poor prognosis of patients with prostate or breast cancers (9, 10), aggressiveness of bladder tumors (11), or tumor recurrence of liver cancer (12). Also, many oncoproteins and tumor suppressors such as c-MYC (13), p53 (14), and YAP (15, 16) are O-GlcNAc modified. These observations link O-GlcNAc with multiple hallmarks of cancer, including sustaining proliferative signals and deregulating cellular energetics. Cancer cells are usually metabolically active. In particular they often sustain high rates of glucose uptake, a phenomenon commonly known as the Warburg effect, which is a preferential reliance on aerobic glycolysis to obtain energy (17). How elevated uptake of glucose triggers O-GlcNAc modification of cancer-related proteins and controls their activities is accordingly a topic of growing interest.
Homeodomain-interacting protein kinases (HIPKs; HIPK1–4 in mammals and Hipk in Drosophila [fruit fly]) are protein kinases involved in the regulation of signal transduction (18⇓⇓–21), cell proliferation and differentiation (22, 23), apoptosis (24), stress response (25), embryonic development (26), angiogenesis (27), adipogenesis (28), as well as immune homeostasis (29). The activity of HIPK2 (the most studied member of the mammalian HIPK family) is governed by multiple strategies depending on the environments. HIPK2 is normally maintained at low levels by proteasomal degradation involving various ubiquitin E3 ligases such as Siah1 and Siah2 (30, 31). During hypoxia, HIPK2 degradation is facilitated by the increased association between HIPK2 and Siah2 (31). DNA damage, on the other hand, disrupts the HIPK2–Siah1 interaction, protecting HIPK2 from degradation (30). Furthermore, high oxidative stresses modulate HIPK2 SUMOylation and acetylation states, which influence HIPK2 localization (32). Given the capacity for HIPK2 to respond to environmental cues and the growing recognition that nutrient sensing through OGT is a key regulator of cellular homeostasis, we were intrigued by the potential for O-GlcNAc modification to serve as a nutritional regulator of HIPK(s).
Our previous work has established that overexpression of Hipk in Drosophila promotes tissue growth abnormalities and several tumor-like features, including metabolic reprogramming, cell invasion-like behaviors, and cellular changes reminiscent of epithelial-to-mesenchymal transition, including up-regulation of Twist and Matrix metalloproteinase 1 (MMP1) and down-regulation of E-cadherin (19, 33, 34). Using this in vivo model, we show that OGT is not only necessary for Hipk-mediated tissue growth abnormalities, but also sufficient to synergize with mild levels of Hipk to produce tumor-like phenotypes. Furthermore, we find that HIPKs (both fly Hipk and human HIPK2) are O-GlcNAc-modified proteins. Elevated OGT results in the buildup of HIPK proteins in a posttranscriptional manner. In particular, we identify that HIPK2 O-GlcNAcylation at residues S852, T1009, and S1147 are responsible for OGT-mediated stabilization.
High Dietary Sugar Potentiates Hipk-Induced Growth Abnormalities through HBP and OGT.
In Drosophila, Hipk promotes tissue growth in a dose-dependent manner (19). Using the Gal4-UAS system (35) in flies grown at different temperatures, which affect the extent of Gal4-driven transgene expression, we can generate a range of Hipk overexpression phenotypes that can be used to examine genetic and environmental interactions with Hipk (SI Appendix, Fig. S1). Using the dpp-Gal4 driver to induce UAS-hipk expression in a subset of cells in larval leg imaginal discs that give rise to adult leg appendages, we observed distinct effects at 25 °C or 29 °C. Compared with control flies (SI Appendix, Fig. S1A), flies overexpressing hipk at 25 °C (full genotype: dpp-Gal4 > UAS-hipk, abbreviated as dpp > hipk) usually displayed a wild-type (WT) slender leg phenotype (Fig. 1B, white arrowheads and SI Appendix, Fig. S1B) and rarely had one or two mildly disfigured legs (Fig. 1B, black arrowhead, Fig. 1E, and SI Appendix, Fig. S1B). We define this phenotype as mild leg malformation (SI Appendix, Fig. S1 G, Middle). In contrast, the same genotype grown at 29 °C led to a severe leg malformation phenotype (SI Appendix, Fig. S1 C and G, Right) characterized by significant distortion of pharate adult legs and loss of segmentation. Because of such severe growth abnormalities, these flies usually failed to eclose from their pupal cases. The same phenotype was seen when overexpressing two copies of Hipk at 25 °C (SI Appendix, Fig. S1D), which is associated with the previously reported cell hyperproliferation phenotypes observed in two other larval imaginal discs, wing and eye-antennal discs (33).
The Hipk phenotype seen at 25 °C indicates that when hipk-overexpressing flies were raised and fed a normal diet (0.15 M sucrose) (Fig. 1B), hipk expression levels remained close to, yet still below, the threshold required to cause significant growth abnormalities. We took advantage of the sensitized background of the mild Hipk overexpression phenotype to interrogate whether dietary sugar alters Hipk function in controlling tissue growth. Strikingly, we found that a high sucrose diet (HSD; 1 M sucrose) enhanced the abnormal leg phenotype in hipk-overexpressing flies such that nearly all legs were malformed (Fig. 1C, black arrowheads and Fig. 1E). Similarly, we observed that eye-antennal discs from HSD-fed hipk-overexpressing larvae showed an enlarged Decapentaplegic (Dpp) domain (marked by Hipk immunoreactivity) (SI Appendix, Fig. S2 C and D), indicative of augmented Hipk activities and enhanced cell proliferation.
To test if dietary sugar might modulate Hipk tissue growth through flux through the HBP, we added glucosamine to the fly media. Glucosamine bypasses the rate-limiting step governed by GFAT enzymes and directly enters the HBP (Fig. 1A) (3). We found that supplementing the normal diet (0.15 M sucrose) with glucosamine (0.1 M glucosamine) also potentiated the mild leg malformation phenotype in hipk-overexpressing flies (Fig. 1D black arrowheads, Fig. 1E) in a similar manner as feeding with a high sucrose diet. Under either feeding conditions, flies without overexpression of hipk did not show any malformed leg phenotypes (SI Appendix, Fig. S2 E–I) or increased cell proliferation (SI Appendix, Fig. S2 A and B).
Next, we used RNA interference (RNAi) to knock down the rate-limiting enzymes in the HBP, GFATs, which are encoded in flies by gfat1 and gfat2. Notably, simultaneous knockdown of gfat1 and gfat2 suppressed the effect of high sugar on Hipk-induced malformed leg phenotypes (Fig. 1 F, G, and J). To examine if OGT downstream of the HBP is required for the stimulatory effect of high sucrose on Hipk-mediated tissue growth phenotypes, we investigated the effects of genetic inhibition of OGT. Using two independent RNAi fly lines, we found that knockdown of ogt (also known as super sex combs (sxc) in flies (36, 37)) partly rescued the leg phenotype seen after high sucrose feeding (Fig. 1 H–J). We confirmed the knockdown efficiency of the RNAi lines used in this study by qRT-PCR, Western blotting, and/or immunofluorescence (IF) analyses (SI Appendix, Fig. S3). Further supporting a link to the HBP and OGT, we also found that simultaneous overexpression of gfat1 and gfat2 (Fig. 2 C and E) or overexpression of ogt in a dpp > hipk genetic background (Fig. 2 D and E) was sufficient to produce leg malformations reminiscent of Hipk hyperactivation (Fig. 2 B and E) even under normal dietary conditions. Together, using the leg malformation phenotypes as a readout for Hipk activity, our genetic interaction studies indicate that dietary sugar promotes Hipk activity at least in part through the HBP and OGT.
OGT synergizes with Hipk to drive tumor-like tissue growth. (A–D) Adult legs of dpp > hipk + GFP (A), dpp > hipk + hipk (B), dpp > hipk + gfat1 + gfat2 (C), and dpp > hipk + ogt (D) flies. White arrowheads mark the WT-like slender leg phenotype. Black arrowheads mark the malformed legs. (Scale bars, 500 μm.) (E) A violin plot showing the malformed leg phenotype of the indicated animals. The letters A–D refer to the flies shown in Fig. 2 A–D. Numbers of flies counted N: (A) 141, (B) 47, (C) 64, and (D) 76. P values shown were calculated using unpaired two-tailed Student's t test. (F–I) Wing discs of dpp > GFP (F), dpp > GFP + hipk (G), dpp > GFP + ogt (H), and dpp > GFP + hipk + ogt (I) larvae. GFP (green) marks the transgene-expressing cells. (J) Eye-antennal discs of dpp > GFP (Left) and dpp > GFP + hipk + ogt (Middle) larvae dissected 6 d after egg laying (AEL). dpp > GFP + hipk + ogt animals experienced an extended larval phase, and their eye disc (dissected 10 d AEL) is shown (Right). GFP (green) marks the transgene-expressing cells. (K–O) Control eye discs (dpp > GFP) stained for PH3 (red in K), CycE (gray in L), MMP1 (gray in M), dMyc (gray in N), and E-cad (gray in O and O′). (P–T) hipk- and ogt-coexpressing eye discs (dpp > GFP + hipk + ogt) stained for PH3 (red in P), CycE (gray in Q), MMP1 (gray in R), dMyc (gray in S), and E-cad (gray in T and T′). DAPI staining for DNA (blue) reveals tissue morphology. Flies were raised at 25 °C with control diet. (Scale bars, 50 μm.)
OGT Synergizes with Hipk to Drive Tumor-Like Tissue Growth.
As stated in the introduction, we previously established an in vivo fly tumor-like model with elevation of hipk (overexpression of two copies of hipk) using wing imaginal discs in larvae raised at 25 °C (33). Given the genetic interactions between Hipk and OGT observed in the adult legs (Figs. 1 H and I and 2D), we next asked whether OGTs play a role in Hipk-mediated tissue growth effects in larval wing and eye discs.
We found that in larval wing imaginal discs at 25 °C, overexpression of either hipk (single copy) (Fig. 2G) or ogt (Fig. 2H) on their own did not cause any abnormal phenotypes. Coexpression of ogt with hipk, however, resulted in profound tissue overgrowth (Fig. 2I), suggesting that they drive tumor-like growth in a synergistic manner. Coexpression of hipk and ogt also resulted in a delay in timing of pupariation (SI Appendix, Fig. S4A), a phenotype commonly seen when tumor burden is present in larvae (38). A comparable synergy was also seen in larval eye-antennal discs (Fig. 2J and SI Appendix, Fig. S4 B–E) and, in the developmentally delayed larvae, hipk and ogt coexpressing tissues grew in size continuously (Fig. 2J). Furthermore, we found that the cells coexpressing hipk and ogt were characterized by an enrichment in phosphohistone H3 (PH3, a mitotic marker), up-regulation of cyclin E (CycE), matrix metalloproteinase 1 (MMP1), and dMyc (MYC in vertebrates), as well as loss of E-cadherin (E-cad) (Fig. 2 K–T and SI Appendix, Fig. S4 B–E), which are all established markers associated with extensive cancerous growth. These data indicate that OGT is capable of augmenting the tumorigenic potential of Hipk even though hipk is mildly overexpressed in this system.
OGT Is Necessary for Hipk-Induced Tissue Growth Abnormalities.
Next, we set out to evaluate the requirement of OGT for Hipk activity using the leg malformation phenotypes. We shifted the fly culture temperature from 25 °C to 29 °C such that Gal4 activity was increased to drive greater Hipk expression to reach a level sufficient to cause severe growth abnormalities (Fig. 3 A and B black arrowheads, SI Appendix, Figs. S1C and S5 A and B). Coexpression of ogt-RNAi with hipk partially rescued the severe leg malformation phenotype caused by excess Hipk as the leg segmentation was partly restored (Fig. 3C white arrowheads, SI Appendix, Fig. S5 C, E, and F). Expression of ogt-RNAi alone did not cause any abnormal leg phenotypes (Fig. 3D and SI Appendix, Fig. S5D).
OGT is necessary for Hipk-induced tissue growth abnormalities. (A–D) Adult legs of dpp > GFP (A), dpp > hipk + GFP (B), dpp > hipk + ogt-RNAi (C), and dpp > ogt-RNAi + GFP (D) flies. Black arrowheads in B mark the legs with severe malformation. White arrowheads in C mark the legs with mild malformation. See the side views and quantifications in SI Appendix, Fig. S5. (Scale bars, 500 μm.) (E–H) Wing discs of dpp > GFP (E), dpp > hipk + GFP (F), dpp > hipk + ogt-RNAi (G), and dpp > ogt-RNAi + GFP (H) larvae. The transgene-expressing domain was marked by GFP (green in E and H; gray in E″ and H″) or Hipk staining (red in F and G; gray in F″ and G″). (I and J) In an OGT mutant background (sxc[6]/sxc[7]), wing discs of dpp > hipk (I) and dpp> GFP (J) larvae are shown. The transgene-expressing domain is marked by Hipk staining (red in I; gray in I″) or GFP (green in J; gray in J″). Flies were raised at 29 °C with control diet. DAPI staining for DNA (blue; gray in E′–J′) reveals tissue morphology. (Scale bars, 50 μm.)
We then examined the requirement of OGT for Hipk-induced tumor-like tissue growth in wing discs. Compared with the control wing discs (Fig. 3E and SI Appendix, Fig. S5F), elevated Hipk (overexpression of one copy of hipk at 29 °C) induced significant abnormal overgrowth (Fig. 3F and SI Appendix, Fig. S5H). When ogt was knocked down in hipk-overexpressing cells, the overgrowth induced by elevated Hipk was largely suppressed, though mild tissue distortions remained (Fig. 3G and SI Appendix, Fig. S5I). We also used ogt mutant alleles, sxc[6] and sxc[7], which possess a point mutation at a splice acceptor site and a nonsense mutation, respectively (36, 37), to generate an ogt null genetic background. Similar to the effects of ogt knockdown, the proliferative potential of the cells with elevated Hipk was largely reduced in the ogt mutant background (Fig. 3I). Altogether, our data illustrate a requirement for OGT in Hipk-induced tissue growth abnormalities, and the requirement is general to all tissues examined.
Depletion of OGT Reduces Hipk Protein Levels.
Given these results, we considered mechanisms by which OGT might influence Hipk activity and found, using immunofluorescence staining experiments, that loss of OGT caused a robust reduction of exogenous Hipk immunoreactivity (Fig. 3 F, G, and I). Compared to a control genetic background (Fig. 4A), fewer hipk-expressing cells were detected and Hipk expression levels were lower in the ogt null background (sxc[6]/sxc[7]) (Fig. 4B) or when ogt was knocked down (SI Appendix, Fig. S6A). Hipk was predominantly nuclear regardless of OGT levels (Fig. 4 A and B and SI Appendix, Fig. S6), indicating that OGT is not involved in the regulation of Hipk subcellular localization. A comparable result was obtained using additional ogt mutant alleles, sxc[4] and sxc[5] (SI Appendix, Fig. S6 B and C), which encode catalytically inactive forms of OGT (OGT-N948I and OGT-Δ1031 to 1059, respectively (36, 37)). We also examined another larval epithelial tissue, salivary glands, because their large cell size facilitates imaging. Depletion of ogt in salivary gland cells caused drastic reductions in Hipk protein levels (Fig. 4 C and D). Using immunoblotting, we confirmed that loss of OGT led to down-regulation of exogenous Hipk protein levels (Fig. 4E and SI Appendix, Fig. S6D). We noted that ogt depletion did not cause loss of dpp-Gal4 expression (Fig. 3 H and J). Thus, ogt depletion does not reduce Hipk protein levels by repressing UAS-hipk transcription. Also, reductions in OGT levels had little effects on the transcriptional levels of endogenous hipk (SI Appendix, Fig. S3B). Taken together, our data imply that the catalytic activity of OGT is required to sustain Hipk protein levels posttranscriptionally within Drosophila.
Depletion of OGT reduces Hipk protein levels. (A and B) Wing discs overexpressing hipk (dpp > hipk) in a WT background (A) or in an OGT mutant background (sxc[6]/sxc[7]) were stained for Hipk using anti-Hipk antibodies (red in A and B; gray in A′ and B′). DAPI was used to stain DNA (blue). (Scale bars, 10 μm.) (C and D) hipk-overexpressing salivary glands without (dpp > hipk + GFP) (C) or with OGT knockdown (dpp > hipk + ogt-RNAi #2) (D) were stained for Hipk using anti-Hipk antibodies (red). DAPI was used to stain DNA (blue). (Scale bars, 50 μm.) (E) Western blot analyses of the exogenous Hipk protein levels in the larval head extracts (which included imaginal discs and salivary glands) obtained from dpp > hipk + GFP, dpp > hipk + ogt-RNAi and dpp > hipk + ogt-RNAi #2 animals. β-Tubulin was used as a loading control. Flies were raised at 29 °C with control diet.
OGT Stabilizes Hipk by Preventing Hipk from Proteasomal Degradation.
Given the dependence of Hipk protein levels on catalytically active OGT, we asked whether OGT controls Hipk protein stability. Using a cycloheximide (CHX) chase assay to observe protein stability in the absence of translation, we found that while Hipk levels dropped slightly in control cells, Hipk levels dropped precipitously when ogt was knocked down (Fig. 5 A and A′). To impair proteasomal degradation, we knocked down rpn2, a component of the 26S proteasome (39), and this restored Hipk protein levels (Fig. 5B) as well as the tumor-like growth in the ogt-depleted background (Fig. 5 C–E). Endogenous Hipk proteins were scarce and hardly detectable (Fig. 5F and SI Appendix, Fig. S7B). However, overexpression of ogt was sufficient to induce accumulation of endogenous Hipk proteins (Fig. 5G and SI Appendix, Fig. S7 C and D). Together, our results suggest that OGT activity positively regulates Hipk protein stability by reducing proteasomal degradation of Hipk.
OGT stabilizes Hipk by preventing Hipk from proteasomal degradation. (A and A′) Dissected larval heads from dpp > hipk + RFP and dpp > hipk + ogt-RNAi larvae were cultured in insect medium and exposed to 200 μg/mL CHX for the indicated times, followed by Western blot analyses of the exogenous Hipk protein levels. β-Tubulin was used as a loading control. Numeric values above the Hipk blot show the relative Hipk protein levels, which are set as 1 at 0 min of CHX treatment. (A′) Quantification of the relative Hipk protein levels of dpp > hipk + RFP (black line) and dpp > hipk + ogt-RNAi (blue line) larval head extracts in the CHX chase assay. Data are mean ± SEM and pooled from three biological replicates. At each time point, the P value shown is calculated using unpaired two-tailed Student's t test. (B) Western blot analyses of the exogenous Hipk protein levels in the larval heads extracted from dpp > hipk + ogt-RNAi and dpp > hipk + ogt-RNAi + rpn2-RNAi animals. β-Tubulin was used as a loading control. (C and D) Wing discs of dpp > hipk + ogt-RNAi (C) and dpp > hipk + ogt-RNAi + rpn2-RNAi (D) larvae are shown. The transgene-expressing domain is marked by Hipk staining (red; gray in C″ and D″). DAPI staining for DNA (blue; gray in C′ and D′) reveals tissue morphology. (Scale bars, 50 μm.) (E) A box and whisker plot showing the ratio of Hipk-positive area to total area of the dpp > hipk + ogt-RNAi wing discs without or with rpn2 knockdown. The letters C and D refer to the fly genotypes shown in Fig. 5 C and D. P value shown is calculated using unpaired two-tailed Student's t test. (F and G) Control (dpp > GFP) (F) and OGT-overexpressing (dpp > ogt) (G) salivary glands were stained for Hipk (red; gray in F′ and G′). DAPI was used to stain DNA (blue). (Scale bars, 50 μm.) Flies were raised at 29 °C with control diet.
OGT Binds to and O-GlcNAcylates Hipk.
To test if Hipk and OGT physically interact with each other, we performed coimmunoprecipitation (co-IP) experiments. Using protein extracts from larval tissues expressing both proteins (dpp > hipk + ogt-flag), we found that Hipk and OGT could be reciprocally coimmunoprecipitated (Fig. 6A). A physical interaction suggested to us that Hipk may be an OGT substrate. To test whether Hipk is O-GlcNAc modified, we employed a chemoenzymatic approach (40) (Fig. 6B). Using a mutant β-1,4-galactosyltransferase (Y289L GalT), we labeled O-GlcNAc-modified proteins with N-azidoacetylgalactosamine (GalNAz). GalNAz-labeled proteins were then biotinylated using click chemistry in combination with a biotin-alkyne probe and precipitated using streptavidin resin. Subsequent immunoblot analyses of the precipitated proteins using antibodies against Hipk revealed marked Hipk O-GlcNAcylation when ogt was coexpressed (Fig. 6B). We validated the selectivity of the ligation chemistry during labeling by showing Hipk was undetectable in the precipitates when GalT was excluded from the chemoenzymatic labeling step (SI Appendix, Fig. S8).
OGT binds to and O-GlcNAcylates Hipk. Flies were raised at 25 °C with control diet (A and B) or the indicated diets (C). (A) Protein lysates extracted from dpp > hipk + ogt-flag larval heads were incubated with either IgG or anti-Hipk antibodies, followed by co-IP. Western blot analyses using anti-Hipk and anti-Flag antibodies showing that flag-tagged OGT coimmunoprecipitated with Hipk proteins (Left). Reciprocal IP showing that Hipk coimmunoprecipitated with flag-tagged OGT (Right). (B) Detection of O-GlcNAc modified Hipk by chemoenzymatic GalT labeling (schematic diagram shown on the Left). Protein lysates extracted from dpp > hipk and dpp> hipk + ogt larval heads were incubated with UDP-GalNAz and the enzyme Y289L GalT, followed by coupling with a biotin-alkyne probe by click chemistry, streptavidin (Strvn) pull-down, and Western blot analyses. Actin was used as a loading control. (C) Detection of O-GlcNAc–modified Hipk by GalNAz feeding and GlcNAz labeling (schematic diagram shown on the Left). Click chemistry was performed in protein lysates extracted from mock-fed or GalNAz-fed dpp> hipk + ogt larvae, followed by streptavidin (Strvn) pull-down and Western blot analyses. Actin was used as a loading control.
To corroborate the GalT labeling result, we adopted a metabolic feeding strategy (Fig. 6C) (41). Larvae coexpressing hipk and ogt were raised on food supplemented with either DMSO (control) or Ac4GalNAz. After feeding, the precursor Ac4GalNAz is converted in vivo through metabolism into UDP-GlcNAz (42). As UDP-GlcNAz is a close analog of UDP-GlcNAc, OGT can use this substrate to transfer GlcNAz onto proteins, to generate O-GlcNAz (42, 43). O-GlcNAz–modified proteins in the larval extracts were then chemoselectively coupled with a biotin-alkyne probe. Streptavidin pull-down followed by immunoblot analyses showed that Hipk was only present among the proteins precipitated from GalNAz-fed animals (Fig. 6C), further supporting Hipk being a substrate for OGT and being O-GlcNAc modified in vivo. Thus, our biochemical analyses confirm that fly Hipk is an O-GlcNAc–modified protein.
HIPK2 O-GlcNAcylation by OGT Promotes HIPK2 Protein Stability.
Our observations made using Drosophila indicate that Hipk is a direct target protein for OGT and that OGT is required to modulate Hipk protein stability and Hipk-induced tissue growth abnormalities. To see whether the regulation of Hipk by OGT is conserved across species, we extended our analyses to human HIPK2 using cultured mammalian cells.
We transfected human embryonic kidney (HEK) 293 cells with OGT and found accumulation of exogenous HIPK2 proteins (Fig. 7A). Comparable results were obtained in MCF7 breast adenocarcinoma cells and mouse embryonic fibroblasts (MEFs). Next, using an MEF cell line where OGT knockout can be induced by administration of 4-hydroxytamoxifen (4HT) (44), we found that loss of OGT correlated with loss of HIPK2 (Fig. 7B). Notably, no statistically significant changes in endogenous HIPK2 mRNA levels were found in HEK293 and MEF cells upon genetic manipulation of OGT levels (SI Appendix, Fig. S9 A and C). In MCF7 cells, however, OGT induced HIPK2 transcriptional up-regulation (SI Appendix, Fig. S9B). Together, our data suggest that, whereas OGT generally promotes HIPK2 protein buildup posttranscriptionally, the transcriptional response of HIPK2 to OGT seems to be cell-type specific.
HIPK2 O-GlcNAcylation by OGT promotes HIPK2 protein stability. (A) Increasing amounts of myc-tagged OGT were cotransfected with myc-tagged HIPK2 in HEK293, MCF7, and MEF cell lines. Forty-eight hours after transfection, protein levels of myc-tagged HIPK2 and OGT were analyzed by Western blotting using anti-myc antibodies. β-Tubulin was used as a loading control. (B) Myc-tagged HIPK2 was transfected in mock (methanol)-treated or 4-hydroxytamoxifen (4HT)-treated MEF cells (an OGT inducible knockout [iKO] strain). Forty-eight hours after transfection, protein levels of myc-tagged HIPK2 were analyzed by Western blotting using anti-myc antibodies. OGT knockout efficiency was evaluated by examining the endogenous OGT levels. β-Tubulin was used as a loading control. (C) Mass spectrometry analyses revealed three O-GlcNAc–modified peptides of HIPK2. Schematic diagram of the human HIPK2 protein (amino acids 1 to 1,198) with its domains. KD, kinase domain; HID, homeobox-interacting domain; PEST, proline, glutamic acid, serine, and threonine-rich domain; YH, tyrosine, histidine-rich domain (also known as auto-inhibitory domain [AID]). (D) WT or alanine mutant variants of myc-tagged HIPK2 were transfected in HEK293 cells. Twenty-four hours after transfection, indicated cells were treated with MG132. Forty-eight hours after transfection, protein levels of myc-tagged HIPK2 were analyzed by Western blotting using anti-myc antibodies. β-Tubulin was used as a loading control. (E) Detection of O-GlcNAc levels of HIPK2. HEK293 cells were transfected with WT or alanine mutant variants of myc-tagged HIPK2 and treated with Thiamet-G. O-GlcNAc–modified proteins were pulled down by WGA-conjugated beads. Myc-tagged HIPK2 in the precipitates and in the input were analyzed by Western blotting. β-Tubulin was used as a loading control. (F and F′) WT or alanine mutant variants of myc-tagged HIPK2 were cotransfected with mock or myc-tagged OGT as indicated. Twenty-eight hours after transfection, protein levels of myc-tagged HIPK2 and OGT were analyzed by Western blotting using anti-myc antibodies. β-Tubulin was used as a loading control. (F′) A box and whisker plot showing the relative protein levels of the WT or alanine mutant variants of myc-tagged HIPK2 in the presence of myc-OGT cotransfection. Data are pooled from three biological replicates. P values showing the significant differences between the WT and the mutant protein levels were calculated using unpaired two-tailed Student's t test.
We next mapped the positions of O-GlcNAc modification on HIPK2 purified from transfected HEK293 cells, which were treated with Thiamet-G, an OGA selective inhibitor (45), to stabilize O-GlcNAc modifications and facilitate mapping sites of modification. By using higher energy collision dissociation (HCD) product-ion triggered electron transfer/higher energy collision dissociation (EThcD) tandem mass spectrometry (MS/MS) analyses, we identified three O-GlcNAc-modified peptides containing five putative OGT target sites (S852, T853, S1008, T1009, and S1147) (Fig. 7C and SI Appendix, Fig. S10). Subsequent analyses confirmed that S852, T1009, and S1147 are O-GlcNAc modified (SI Appendix, Fig. S10 B–D). All of the sites identified are located outside of the kinase domain. S852 and T853 are within a conserved PEST region implicated in protein stability and S1147 within the YH protein interaction domain (sometimes referred to as an autoinhibitory region) (46). Notably, these sites are conserved among mammals (SI Appendix, Fig. S11).
To assess the functional relevance of HIPK2 O-GlcNAcylation, we replaced these serine and threonine residues with alanine by site-directed mutagenesis. Since we could not definitively rule out that all five residues are targets of OGT, we mutated the three confirmed (S852, T1009, and S1147) and two adjacent sites (T853 and S1008) to alanine to address the possibility of these sites being modified. Individual single alanine mutants and a quintuplet alanine (5A) mutant were generated (SI Appendix, Fig. S12). We found that while WT HIPK2 was weakly expressed (Fig. 7D, lane 1), the mutants were expressed below detectable limits (Fig. 7D, lanes 2 to 7). Even in the presence of MG132 (used to impair proteasomal degradation), the mutants were expressed at reduced levels when compared with WT HIPK2 (Fig. 7D, lanes 8 to 14).
To determine the O-GlcNAc states of WT HIPK2 and the mutants, we used higher concentrations of plasmids for transfection, combined with Thiamet-G treatment to prevent the removal of O-GlcNAc modifications (Fig. 7E). Using wheat germ agglutinin (WGA) to selectively pull down O-GlcNAc–modified proteins, we found that WT HIPK2, unlike the alanine mutants, was readily detectable in the precipitates (Fig. 7E), indicating that the serine and threonine residues identified by MS can be O-GlcNAc modified and losses of these residues rendered HIPK2 more resistant to O-GlcNAcylation.
If the O-GlcNAc sites identified are responsible for maintaining HIPK2 protein stability through OGT, we would expect the mutants to be less responsive to OGT-mediated stabilization. Strikingly, OGT overexpression had much reduced effects on the stabilization of the mutants when compared with WT HIPK2 (Fig. 7F, even-numbered lanes, Fig. 7F′). Nevertheless, all mutant variants, like WT HIPK2, could be stabilized to some extent by OGT (Fig. 7F, compare even- with odd-numbered lanes), suggesting the presence of additional, not yet identified, OGT-mediated mechanisms contributing to HIPK2 stability. These data point to the importance of direct O-GlcNAcylation of HIPK2 in the positive regulation of HIPK2 protein stability by OGT.
Cancer cells often vigorously consume glucose, aided by metabolic reprogramming involving multiple processes, for example, elevated expression of glucose transporters (17). Notably, an ample supply of glucose not only facilitates aerobic glycolysis within tumors, but also drives increased flux through parallel pathways, including the HBP (47). As a consequence, increased levels of UDP-GlcNAc and subsequent hyper–O-GlcNAcylation are found in nearly all cancers examined, including breast, prostate, colon, lung, liver, pancreatic, and leukemias (48). Indeed, global O-GlcNAcylation is emerging as a node linking glucose availability and cancer progression (48).
The link between dietary sugar and increased tumorigenesis is found to be conserved in Drosophila (49⇓–51). Using various fly cancer models, researchers have shown that excess sugar intake affects cancer progression through multiple pathways. For example, high dietary sugar has been shown to drive the malignancy of Ras/Src tumors and neoplasia of EGFR-driven tumors through evasion of insulin resistance and lactate dehydrogenase-dependent aerobic glycolysis, respectively (49⇓–51). The involvement of hexosamine signaling in fly tumorigenesis, however, has not been explored. Here we report that the activity of Hipk, a proliferation-promoting protein in flies, is controlled by the HBP–OGT axis in response to metabolic nutrients (Fig. 1). Using the fly Hipk tumor-like model, we demonstrate that OGT is both necessary and sufficient for Hipk-induced growth abnormalities (Figs. 2 and 3). Thus, our work defines the HBP–OGT axis as a glucose-dependent mechanism regulating Hipk-mediated growth control. This is consistent with several studies of O-GlcNAc–dependent regulation of growth-promoting proteins such as YAP in mammals (15, 16).
In addition, we observed a mild up-regulation of OGT in the Hipk-overexpressing cells (SI Appendix, Fig. S5H), inferring that OGT may be a downstream target of Hipk and that the up-regulation of OGT may sustain ectopic Hipk expression and form a positive feedback loop. Recently, we reported that fly Hipk tumor-like cells display elevated aerobic glycolysis driven by up-regulation of dMyc (MYC in vertebrates) (34). It is therefore tempting to speculate that the increased glucose metabolism could stimulate HBP flux and generate more UDP-GlcNAc for O-GlcNAcylation, and together with the mild OGT up-regulation, the combined effects may ultimately feedforward to sustain high Hipk protein levels and reinforce tumor-like proliferation. Future studies may shed light on this mechanism.
Although Hipk proteins are proposed to be maintained at low levels through degradation by the proteasomal machinery, minimal amounts of Hipk are indispensable for normal growth and development as flies homozygous mutant for hipk are not viable (21). This hints at the existence of cellular mechanisms that oppose Hipk protein degradation. Our work demonstrates that OGT stabilizes both endogenous and exogenous Hipk and protects Hipk from proteasomal degradation (Figs. 4 and 5). We confirm that fly Hipk is an O-GlcNAc–modified protein (Fig. 6). While the fly Hipk O-GlcNAc motifs and their roles remain unknown, we show that OGT-mediated stabilization of human HIPK2 relies on direct O-GlcNAcylation of HIPK2 at residues S852/T853, S1008/T1009, and S1147 (Fig. 7). This report describes HIPK2 O-GlcNAcylation as a molecular mechanism that controls HIPK2 protein stability. Given the nutrient-sensing nature of O-GlcNAc, it is conceivable that HIPK2 O-GlcNAcylation might contribute to the nutrient sensitivity of HIPK2 abundance. Intriguingly, a study shows that HIPK2 protein levels increased in a mouse model of diabetes, and the authors attributed the effect to down-regulation of the E3 ubiquitin ligase Siah-1 (52). Hence, the nutritional regulation of HIPK2 might be achieved by multiple strategies like direct O-GlcNAcylation and impairment of proteasome system, or their cooperative effects.
In summary, our work illustrates the conserved regulation of HIPK (fly Hipk and human HIPK2) protein stability by OGT and demonstrates the functional consequences of this regulation in tumor-like events using an in vivo fly model. The roles of human HIPK2 in cancers are context dependent and yet to be fully understood. On the one hand, HIPK2 activates the tumor suppressor p53 after UV irradiation (53, 24). Also, HIPK2 acts as a tumor suppressor upon induction of two-stage skin carcinogenesis (54) or γ-radiation–induced tumorigenesis (55). On the other hand, HIPK2 promotes cytoprotection in cancer cells when challenged with chemotherapeutic drugs (25). Elevation of HIPK2 is also associated with malignancy of pilocytic astrocytomas (56) and cervical carcinogenesis (57). These results raise concerns that targeting HIPK2 may have paradoxical effects. Elevated HIPK2 is also implicated in fibrosis in kidney (58), lung (59), and liver (60). Thus, depending on the context, we believe that the dynamic control of HIPK2 abundance by O-GlcNAc modification reported in our study can be of great interest and exploited in the treatment of HIPK2-related disorders with the use of dietary control or metabolic drugs targeting the HBP–OGT axis.
Fly Culture.
Crosses were kept at 25 °C or 29 °C as indicated on the food. Food recipes and fly strains are provided in SI Appendix, SI Materials and Methods.
Immunofluorescence Microscopy.
Larval imaginal discs or salivary glands were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, followed by standard immunofluorescence staining protocol. Details are provided in SI Appendix, SI Materials and Methods.
Quantitative Real Time-PCR.
Total RNA isolation was performed using the RNeasy Mini Kit (Qiagen 74104), followed by first-strand cDNA synthesis using the PrimeScript Kit (Clontech 6110A). qRT-PCR was performed using the SensiFAST SYBR Kit (Bioline 92005) on the StepOne Real-Time PCR System (Applied Biosystems). See SI Appendix, Table S1 for primers used in this study.
Western Blotting.
Cells or tissues were lysed with 1× Cell Lysis Buffer (Cell Signaling Technology), supplemented with 1× Protease Inhibitors (Roche) and 1 mM phenylmethylsulfonyl fluoride. Protein lysates with 1× SDS sample buffer were resolved on SDS/PAGE and then transferred to nitrocellulose membranes. Membranes were blocked with 5% skimmed milk or 5% bovine serum albumin before primary and secondary antibody incubation. Images were acquired by a FujiFilm LAS-4000 Chemiluminescent Scanner or the Li-Cor Odyssey Imaging System. A list of antibodies is provided in SI Appendix, SI Materials and Methods.
Cycloheximide (CHX) Chase Assay.
Wandering third-instar larvae were rinsed in PBS and dissected. The anterior half was cultured in insect medium (HyClone) supplemented with CHX (Sigma, 200 µg/mL) for 0 to 120 (or 150) min at room temperature. Tissues were then homogenized in lysis buffer by brief sonication and used for Western blot analyses.
Coimmunoprecipitation and Detection of Hipk and HIPK2 O-GlcNAcylation.
Protein extracts were incubated with indicated antibodies at 4 °C overnight, followed by incubation with a mixture of protein A (Invitrogen 10002D) and protein G (NEB S1430S) magnetic beads for 3 h at 4 °C. The beads were washed with lysis buffer and boiled in 3× SDS sample buffer. The supernatants were then analyzed by Western blotting. Details of WGA pull down, GalNAz feeding, and GalT labeling for detection of Hipk or HIPK2 O-GlcNAcylation are provided in SI Appendix, SI Materials and Methods.
Cell Lines and Cell Culture.
HEK293 and MCF7 were cultured in Dulbecco's modified eagle medium (DMEM) high glucose (25 mM glucose) (Gibco 11965-092) supplemented with 10% fetal bovine serum (FBS). All cells were cultured in a humidified, 5% CO2 incubator at 37 °C. Cells were transiently transfected with mock (empty vector) or indicated plasmids using Lipofectamine 3000 (Invitrogen) or TransIT-2020 (Mirus Bio) according to the manufacturers' instructions. Cells were harvested 48 h after transfection unless otherwise indicated. pcDNA3.1-Myc-HIPK2 and pcDNA3.1-Myc-HIPK2-S1008A were purchased from GenScript. pCMV-Myc-OGT-WT has been described (61).
MEF cells containing lentivirus encoding mutated estrogen receptor (mER)-Cre-2A-GFP construct were described previously (44). Cells were cultured in DMEM (high glucose, Gibco) with 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin at 37 °C in a water-jacketed, humidified CO2 (5%) incubator. Cells were plated at 30 to 50% confluency. Unless otherwise noted, Cre-recombinase was activated to excise OGT through incubation with 2 µM 4-hydroxytamoxifen (4HT in methanol, Bioshop) for 72 h after plating. Fresh medium and 4HT were changed every 24 h.
Site-Directed Mutagenesis.
For single-site directed mutagenesis, PCRs were run for 18 cycles of 15 s at 95 °C, 30 s at 60 °C, and 18 min at 72 °C using Q5 High-Fidelity DNA Polymerase (NEB M0491). In addition to GC enhancer, 3.5% DMSO was included in the PCR mix because of the GC-rich regions in the HIPK2 insert. The PCR products were treated with FastDigest DpnI (Thermo Scientific FD1704) to remove the template DNA, followed by transformation into One-Shot MAX Efficiency DH5α-T1 Competent Cells (Invitrogen 12297016). Multisite-directed mutagenesis was performed using the GeneArt Site-Directed Mutagenesis PLUS System (Invitrogen A14604) with slight modifications. DNA methylation was not included prior to PCR. Instead, template DNA was removed using DpnI digestion, followed by in vitro recombination reaction. See SI Appendix, Table S2 for the mutagenic primers used. Mutations were then verified by DNA sequencing.
Mapping of O-GlcNAc Sites.
HEK293 cells were transfected with pcDNA3.1-Myc-HIPK2, followed by treatment with Thiamet-G to block the removal of physiological relevant O-GlcNAc attachments. HIPK2 harvested from the cells was purified through immunoprecipitation using anti-Myc tag antibodies and succinylated wheat germ agglutinin (sWGA) enrichment. The resulting purified HIPK2 was run on gels, followed by in-gel tryptic digestion and then LC-MS/MS. Data were obtained from two biological samples. Details for in-gel digestion and MS to map the O-GlcNAcylation sites of HIPK2 are provided in SI Appendix, SI Materials and Methods.
The data were represented by mean ± SEM. The P values were determined by two-tailed Student's t tests. P < 0.05 was considered statistically significant. Bar graphs were generated by Microsoft Excel. Violin plots and box and whisker plots to represent the distribution of data were generated by BoxPlotR (62).
We are grateful to the Developmental Studies Hybridoma Bank, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, and Jackson ImmunoResearch Laboratories for providing fly strains and antibodies. We thank M. Alteen for production of chemical materials. We appreciate all the discussion and input from our laboratory's current and past members, including Dr. G. Tettweiler, Dr. E. Hall, S. Kinsey, L. Madonsela, R. Tse, T. Wiens, L. Zhe, J. Blaquiere, N. Wray, and E. Hoesing. We also thank J. Blaquiere and R. Chung for conducting a genetic screen to search for Hipk modifiers. This work was funded by operating grants from the Canadian Institutes of Health Research, PJT-156204 and PJT-148732. Mass spectrometry data were acquired at the Academia Sinica Common Mass Spectrometry Facilities for Proteomics and Protein Modification Analysis located at the Institute of Biological Chemistry, Academia Sinica, supported by Academia Sinica Core Facility and Innovative Instrument Project (AS-CFII-108-107).
↵1Present address: Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
↵2To whom correspondence may be addressed. Email: everheye{at}sfu.ca.
Author contributions: K.K.L.W., T.-W.L., Y.-Y.C., K.-H.K., D.J.V., and E.M.V. designed research; K.K.L.W., T.-W.L., J.M.P., and Y.-Y.C. performed research; D.A.R.S. contributed new reagents/analytic tools; K.K.L.W., T.-W.L., J.M.P., D.A.R.S., Y.-Y.C., K.-H.K., D.J.V., and E.M.V. analyzed data; and K.K.L.W., T.-W.L., D.J.V., and E.M.V. wrote the paper.
This article is a PNAS Direct Submission. J.A.H. is a guest editor invited by the Editorial Board.
Data deposition: Data and materials are available on the Open Science Framework at https://osf.io/epkmj/.
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J. Rappsilber,
M. Tyers
, BoxPlotR: A web tool for generation of box plots. Nat. Methods 11, 121–122 (2014).
You are going to email the following The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila
Kenneth Kin Lam Wong, Ta-Wei Liu, Jessica M. Parker, Donald A. R. Sinclair, Yi-Yun Chen, Kay-Hooi Khoo, David J. Vocadlo, Esther M. Verheyen
Proceedings of the National Academy of Sciences Jan 2020, 201912894; DOI: 10.1073/pnas.1912894117


Background – Claire McWhite's Site – projects, etc.
Attached is our Letter to the Editor describing our major concerns with the data analysis in srep37855 and srep39328. These papers use quantitative proteomics to identify protein expression differences in response to 1) maize genetic modification and 2) glyphosate ingestion in rats, respectively.
However, we found serious flaws in the methods applied, which render the analysis technically unsound. The methods deviate substantially from standards adopted by the field. Particularly, if any single peptide from a protein was found differently across samples, even just a single observation, the entire protein was counted as differentially expressed. We detail more methodological errors in the attached letter.
Additionally, though requested, the authors have not provided the unprocessed mass spectrometry files required to reproduce their analysis.
Text from their response is in red.
Both of these publications actually quantify peptides, and then misleadingly claim to have quantified proteins. Quantification at the peptide-level is not statistically equivalent to quantification at the protein-level. Using single peptides to make conclusions about protein level while discarding information from other peptides from the same protein necessarily leads to false-positives, spurious conclusions, high noise, and lack of power.
They are essentially peptidomic analyses falsely presented as proteomic analyses. To our knowledge, no one has ever done this in a publication before. The authors claim to use conventional methods, but this idea is refuted by their own support citations (Ashton 2015, Höttlä 2015) and multiple other recent TMT protein quantification papers that correctly combine measurements from all peptides that map to a protein sequence to quantify proteins.
Our position is that both of these papers are profoundly flawed and erroneous.
Any conclusions drawn from the analysis described as protein quantification in these papers are unsupported and not scientifically sound.
Any future paper that attempts to use the same quantification method will also be flawed and unreliable.
Received response to our letter to the editor from Michael Antoniou et al. Will not be uploaded here after permission to post asked and denied by Dr. Antoniou.
These are our materials related to the project from before we had access to the .RAW machine files that were necessary to complete a complete reanalysis. These analyses and letters are based on a forensic data reanalysis working backwards from data originally provided with the paper.
Our interest is in maintaining rigor and standards in proteomics methods.
I'm a Cell and Molecular Biology PhD student in the Marcotte Lab at the University of Texas at Austin, with the Center for Systems and Synthetic Biology.


text:
import Mongoose, { Schema } from 'mongoose';
import bcrypt from 'bcrypt-nodejs';

const UserSchema = new Schema({
  email: String,
  password: String
});

UserSchema.methods.toJSON = (newUser) => {
  const user = newUser.toObject();
  // Removing password from returned User model, so we won't send it back to view.
  Reflect.deleteProperty(user, 'password');
  return user;
};

UserSchema.methods.comparePasswords = (enteredPassword, fromDbPassword, callback) => {
  bcrypt.compare(enteredPassword, fromDbPassword, callback);
};

UserSchema.pre('save', function(next) {
  bcrypt.genSalt(10, (err, salt) => {
    if(err) return next(err);
    bcrypt.hash(this.password, salt, null, (err2, hash) => {
      if(err2) return next(err2);
      this.password = hash;
      console.log(hash);
      next();
    });
  });
});

export default Mongoose.model('User', UserSchema);

redpajama_set_name: RedPajamaGithub


Based in Winnipeg, Manitoba we have over 100 years of experience in the monument and granite industry. We provide a range of products from memorials to benches to plaques and everything in between.
Please feel free to tour our site, and if you have any questions or comments don't hesitate to contact us for more information.


home › posts › 7 Facts About Sigillaria, O... ten trees planted for every item purchased.
We hear a lot about the growing list of extinct animals, but plants do also go extinct. Currently, more than 1,100 trees are listed as critically endangered. But many other trees have gone extinct for reasons not caused by humans. Among the most interesting trees to ever live is the Sigillaria. Here are 7 facts about Sigillaria.
If you go out into the woods today, you won't see any Sigillaria growing wild. Fossil records indicate that Sigillaria evolved during the Late Carboniferous period, but eventually went extinct during the Permian period. In total, the tree existed for around 100 million years, but went extinct around 383 million years ago.
Perhaps one of the strangest things about Sigillaria was its bark. Instead of having bark like today's trees, the Sigillaria was protected by a series of diamond-shaped scales. The tree's base was not comprised of wood like today's trees, but densely packed leaf bases.
For an ancient plant that wasn't made of wood, Sigillaria grew to be pretty tall. In total, Sigillaria could grow to 30 meters, or just shy of 100 feet. Sigillaria reached its full height fairly rapidly.
Ancient lycopods had a tendency to grow quickly and live short lifespans. Sigillaria was no exception. They reached their maturity in as little as just a few years. Some scientists believe that Sigillaria died after it reproduced, but currently, that is only a theory. There is no supporting science.
If you traveled back in time and saw a Sigillaria, you might think it somewhat resembles a palm tree. Sigillaria had long, slender grass-like leaves that grew from a fork at the top of the tree. These leaves attached directly to the stem of the tree. Sigillaria did not grow branches like today's trees do.
Most of today's trees reproduce by creating seeds, but Sigillaria did things a little differently. Instead of producing seeds, Sigillaria reproduced by creating spores. It would grow structures that slightly resembled today's pinecones that attached to the trunk of the tree. These cones would open and disperse the spores.
Sigillaria was related to plants falling into the Division Lycopodiophyta. Plants belonging to Lycopodiophyta reproduce by spores, just like the Sigillaria did. There are more than 1,290 species that still live today that belong to this classification.


"Control the standard by the details, show the power by quality". Our business has strived to establish a highly efficient and stable team staff and explored an effective good quality regulate course of action for 4 Pcs Packed Gel Pens , Blister Card Packed Gel Pens , 12 Colors Gel Pens , the trust is getting there. Our enterprise normally at your provider anytime.
"We're going to devote ourselves to offering our esteemed shoppers with the most enthusiastically thoughtful expert services for 4 Pcs Packed Gel Pens , Blister Card Packed Gel Pens , 12 Colors Gel Pens , We aim to build a famous brand which can influence a certain group of people and light up the whole world. We want our staff to realize self-reliance then achieve financial freedom lastly obtain time and spiritual freedom. We do not focus on how much fortune we can make instead we aim to obtain high reputation and be recognized for our products. As a result our happiness comes from our clients satisfaction rather than how much money we earn. Ours team will do best for you always.


text:
France, Germany call for a change of European regulatory rules
By Claire Stam | EURACTIV.com
Altmaier's decision to actively shape industrial policy together with France can be seen as a move to protect Germany's 'Mittelstand'.
Less than two weeks after the European Commission blocked the merger of Siemens and Alstom, France and Germany published a joint industrial manifesto on Tuesday (19 February) calling for the EU merger rules to be changed.
"Today, amongst the top 40 biggest companies in the world, only five are European," the manifesto reads.
At a joint press conference in Berlin on Tuesday, French Minister of the Economy and Finance, Bruno Le Maire, and German Economy Minister Peter Altmaier both underlined what they termed a "massive disadvantage" of European companies in comparison to their Chinese and American counterparts.
"In order to be able to create a new European champion, it is necessary to change the European regulatory framework," Le Maire said while emphasising that countries like China heavily subsidise their own companies.
France and Germany prepare plan to create European champions
Following the rejection of the Alstom-Siemens merger, France and Germany are working on a review of the EU's competition rules to create European champions capable of competing with US and Chinese multinationals.
The two European countries suggested updating merger guidelines to take greater account of competition at the global rather than the European level.
In their five-page strategy paper, France and Germany also proposed giving the European Council, the forum representing member states, the right to override some antitrust decisions by the European Commission in certain "well-defined cases".
"It's the first time that France and Germany have put forward proposals to transform EU competition rules," Le Maire said. "We have powerful, modern technologies and we don't want them to serve other continents than ours."
Speaking at a political event later on Tuesday, Le Maire called the European Commission's refusal to let Siemens and Alstom merge an economic mistake and a political flaw.
Six takeaways from Siemens-Alstom rejection
The European Commission decided on Wednesday (6 February) to block the merger of Siemens and Alstom, meant to create a European champion in the railway sector, due to the negative impact it would have on the European market and consumers.
"Sometimes there is nothing more stupid than European rules," he said.
Alongside a change of the regulatory rules, the French-German industrial manifesto also calls for "massive investment in innovation" as well as measures to protect European technologies, companies, and markets.
Both ministers emphasised that completing the Capital Markets Union will enable European financial markets to support innovation in the industry.
"It's all about keeping the created value in Europe," Le Maire said.
"You don't want to see a company that has been funded for many years gone into the hand of investors," he added, referring to the 2016 purchase of German robotics maker Kuka by China's Midea, a move that created much controversy in Germany.
In this respect, Altmaier's decision to actively shape industrial policy together with France can be seen as a move to protect Germany's 'Mittelstand,' the backbone of the country's economy, and a clear break with its traditional "hands off" approach to business and market decisions.
New joint battery initiative
Both ministers also presented their new joint initiative where Germany and France earmarked a combined €1.7 billion to support the local production of battery cells for electric vehicles.
"I expect a decision about the consortia very soon, probably in the coming weeks," Altmaier said, adding that France and Germany hoped to get European Commission state aid approval before 1 April.
Both ministers declined to say where the first plant will be and instead stressed that this project is open to all member states.
"So far, Poland, Spain and Italy have expressed their interest in the project," Le Maire said.
The push is meant to reduce European carmakers' dependence on Asian electric vehicle battery suppliers and protect local jobs, at risk from the shift away from combustion engines.
"It is dangerous to depend on foreign suppliers, particularly from Asia," Le Maire said.
"What we want, is to keep the whole value chain European,2 he added.
In his speech, the French minister called China's new silk road the most important strategic project worldwide.
"With its silk road project, China is about to take it all (on the economic world stage)," he said.
[Edited by Zoran Radosavljevic]
One response to "France, Germany call for a change of European regulatory rules"
Julian Stoev says:
SpaceX is not the biggest company, but it basically took over the market owned previously by giants in Russia, EU and US. Tesla is also very small compared to others. Amazon was also small long time ago. … Size has nothing to do with innovation . In fact the relationship is probably reverse. EU companies also receive huge subsidies, not only China and US. And this did not save Europe from miscalculation like A380. My impression is that the idea is to create "too big to fail " companies and continue to poor socialist money from the common EU budget on them.

redpajama_set_name: RedPajamaCommonCrawl


Most photos on my site require Registration and a Private Link to view. REGISTRATION REQUIRES A VALID EMAIL ADDRESS TO AUTOMATICALLY BOUNCE BACK YOUR PERSONAL LINK TO COMPLETE REGISTRATION. Looking for an event/game? Email me at don@buyyourpic.com with Date, Details, and a brief description to be eligible for your own private link. Not all events/games are posted online or available.
I shoot photos for the ScoreBoard Magazine plus some freelance/contract work. I am a member of the National Association of Press Photographers ( http://www.nppa.org ) and a Sports Shooter Member ( http://www.SportsShooter.com ). SportsShooter.com is an online community and resource for sports photographers and other working photojournalists. Membership is by invitation and candidates must submit a portfolio illustrating their capabilities for consideration to become an eligible member.
Want coverage for your athlete or team? Contact me at don@buyyourpic.com for details. Please provide your name, daytime phone number, and short description of coverage requested.
The pictures posted in galleries on BuyYourPic are uploaded without custom color correction since the Professional Lab color corrects each print as ordered to help make it possible for you to receive the best print possible. Need to contact me: Don Bradley email address is don@BuyYourPic.com . Visit my SportsShooter site for more information about me at www.sportsshooter.com/doncbradley . . . . . . . . . . . . . . . I specialize in sports photography, action shots, team and individual packages, as well as on location work for portraits, individuals, families, groups. You are responsible for verifying and adjusting the crop at checkout.


text:
Birds described in 1874 include Island leaf warbler, Bar-bellied pitta, Coppery-naped puffleg, Streak-throated barwing, Red-and-white spinetail,  Papuan hawk-owl, Bay-vented cotinga, Arfak catbird, Rufous-webbed brilliant

Events
Death of Ferdinand Stoliczka

Publications
Spencer Fullerton Baird, Thomas Mayo Brewer , and Robert Ridgway, 1874. A History of North American Birds: Land Birds. Little, Brown, Boston. Volume I, 596 pp.; Volume II, 590 pp.; Volume III, 560 pp. Retrieved 14 January 2013. A special edition, published in the same year, of 50 copies contained 36 plates hand-colored by Ridgway.[110]1873
Henry Haversham Godwin-Austen, Birds of Assam (1870–1878)
Richard Bowdler Sharpe,  Catalogue of the Birds in the British Museum London
Władysław Taczanowski 1874. Description des Oiseaux nouveaux du Pérou central Proceedings of the Zoological Society of London 1874
Etienne Mulsant, Histoire Naturelle des Oiseaux-Mouches, ou Colibris constituant la famille des Trochilides (published 1874-77)
Ongoing events
Theodor von Heuglin Ornithologie von Nordost-Afrika (Ornithology of Northeast Africa) (Cassel, 1869–1875)
John Gould The birds of Asia 1850-83 7 vols. 530 plates, Artists: J. Gould, H. C. Richter, W. Hart and J. Wolf; Lithographers:H. C. Richter and W. Hart
Henry Eeles Dresser and Richard Bowdler Sharpe  A History of the Birds of Europe, Including all the Species Inhabiting the Western Palearctic Region.Taylor & Francis of Fleet Street, London
Paolo Savi Ornitologia Italiana Firenze :Successori Le Monnier,1873-1876. (opera posthuma 1873–1876)
The Ibis

References

Bird
Birding and ornithology by year

redpajama_set_name: RedPajamaWikipedia


The glossy colour magazine familiar to Australian Speedway fans will feature a well-documented review of World Series events and personalities including articles focusing on the popular Speedweek Carnival, our USA based competitors, The Devil and Western Swings plus a full review of WSS31 in the April Edition.
Managing Editor Phil Jordison, an avid speedway historian who has reported on the series since its inception in 1987 is thrilled to be 'on the road' again.
This exciting aspect of the association is an innovative approach to the official QSS World Series Magazine. For the first time Oval Express will include a comprehensive segment dedicated to WSS31. The second half of the magazine will be the official WSS Magazine and will be on sale at your nearest stockiest from mid-December and at QSS WSS31 venues.
WSS31 Co-ordinator Shawn Mortimer said this was a project 18 months in the making and trusts it will be a great record of posterity for all concerned.
Photo: Managing Editor of Oval Express Phil Jordison recently celebrating the 200th edition of Oval Express Speedway Magazine.


Insulin The Insulin sub-category is for discussion about insulin, regardless of the type of diabetes you or your loved-one with diabetes have. Research/Cure The Research/Cure sub-category is for posting and discussion of news about diabetes-related research, including research toward a cure for any type of diabetes. Oral Medications and non-insulin injectables The Oral Medications and Non-Insulin Injectables category is for discussion of any/all diabetes medications except insulin, for both people who do and do not take insulin in addition to other meds.


text:
Spectacular performance of Monteverdi's Orpheus in Mørdrup Kirke
Ole Josephsen, music reviewer (Helsingør Dagblad, 18 September, 2014)
A musical work, created over 400 years ago, can still have a message for modern man. This was proven by the instrumental and vocal ensemble Copenhagen Soloists Sunday afternoon with 15 musicians and 7 singers in Mørdrup Kirke.
The large audience was offered a rare experience, as the world's first opera, Monteverdi's "Orpheus" of 1607 was resurrected in this, Espergærde Music Society's first concert of the new season.
Monteverdi had herewith made scenic the Greek tragedy of Orpheus, who loses his beloved Euridice, wins her back, only to lose her again. It is the story of the hero, who defies the Gods, yet must learn, that such actions cannot go unpunished.
Mathias Hedegaard had the major role as Orpheus, and with his balanced and well-resonating tenor he expressed convincingly the hero's alternating moods, from the euphoric in the joy of love to Euridice, to the dramatic in the anger and grief over having lost her. His prayer to Charon in order to be sailed over to Hades "Possente spirito", was ornamented with finely controlled coleraturas and was no doubt the highlight of the opera.
Christina Larsson Malmberg was a beautiful sounding soprano in the role of Euridice. Anna Jobrant, light soprano, and Simone Rønn, dark alto, proved great virtues in the many roles sung, and in the male roles were Adam Riis, Erlend Tyrmi and Steffen Bruun, deep bass, who completed the spectrum of sound.
The internationally collated instrumental ensemble applied an arsenal of baroque instruments, for example dulcian, cornett, theorbo and gamba, and was well complemented by Mordrup Kirkes acoustics. Under Jonathan Ofir's flaming conducting, the 15 musicians with their heartfelt sense for baroque music were able to create the appropriate accompaniement and counterweight for the singers.
Jonathan Ofir deserves respect for the feat of setting up such a force of singers and players and make it all come together to a complete whole.
We were witness to a spectacular performance of Monteverdi's Orpheus.


If you have a plumbing problem, then you are spoil for choice and should consider hiring the best ones. Here are the criteria you should use while vetting them.
• Consider the specific demands of the job - Some plumbing Everett jobs require specialized attention that only a plumber with a certain qualification can handle. If your toilet is backed up, you should hire a plumber with expertise in that areas and so on.
• References and reputation- They should have a reputation of consistent and top notch customer service. This means that you look at the reviews online and what other people say through word of mouth.
• The reach they have- The plumbing Everett service you should hire should have adequate connections to the local hardware store and get discounts on equipment. This will translate to savings in your wallet.
• Manpower- The plumbing Everett service should display adequate manpower to deal with a job of any scale. Sometimes the job might be very extensive: it could be a full scale flood or a remodel or an overhaul of the entire plumbing system in your house. A one-man show will not be incapable of finishing the task as soon as possible.
When you have a qualified plumbing Everett service in your employ, then you can rest easy because you know they can do any plumbing job for you. Nobody knows when a problem might arise and it is always to be safe than sorry.
Plumbing emergencies might happen at any time and you have to call a plumber Everett.
When you want avoid calling a plumber Everett, you have to avoid creating a situation where you will need to hire one. You can take care of the plumbing around your house, by simply taking care of small things around the house. Below are some the things that you should take care of to avoid making the call to the plumber Everett or keep calling them.
1. If the piping is really old, have it replaced as soon as possible. This will essentially kill two birds with one stone: your house's value will rise significantly and you won't have to constantly call a plumber Everett to fix your house.
2. Loose fixtures should be tightened or replaced if the threads are worn out. If you know how to attach these fixtures, then you can do so with plumbers tape in the threading. Otherwise, you will have to make the call to the plumber to get the issue fixed.
3. Turn off the valve supplying water. When you cut off the source of the problem, you can work to get rid of the problem that you are facing instead of compounding it.
To avoid making emergency calls to a plumber Everett, you will have to schedule a call for maintenance check regularly. It sounds counterproductive when you say out loud but you have to if you want to avoid making that dreaded call during an emergency.
HVAC systems require a lot of care. There are a lot of things that you can ensure that your system runs efficiently. It is important that you take into consideration when it is time to install an new HVAC Chelsea MA system. There comes a time when the bills are too high and you have time and again had repairs done to it but nothing has happened; the system is very inefficient. When it reaches this point, you should consult a HVAC Chelsea MA installation expert to help you address this issue. They will suggest what systems to buy that will guarantee efficiency.
If you have a working system already or are contemplating buying a HVAC Chelsea MA system, you have to know how to take care of it. Here is a brief guide that will ensure your systems longevity.
1. Have your filters replaced every 30 days. This part of the system is very easy to do on your own. These filters can be got from your local hardware store and they are very affordable. When dust accumulates in your system, you will have to replace it because it affects its efficiency.
2. The ducts in the ventilation shaft of your HVAC Chelsea MA system are clear because obstructions make it work doubly hard causing wear and tear on the parts.
3. The refrigerants should be replaced with environmental friendly ones and they should be replaced by a professional.
Plumbing is a major component of a house. It cannot be ignored. If you ignore it, you will end up paying for it dearly. Plumbing emergencies like leakages might render your house uninhabitable. This is where hiring a good plumbing Chelsea MA service will come into play. Sure any plumber can do the job of fixing a problem but it takes a good plumber to ensure that future problems are forestalled as early as possible. You too can take mitigating steps that will ensure your plumbing operates at optimal efficiency and save money at the same time.
1. Hire a good service plumbing Chelsea - A good plumber Chelsea will avert future problems that might arise from wear and tear over the years.
2. Take care of the insulation of the piping - During winter, the most common plumbing Chelsea MA problem you will experience is the bursting of pipes. This can be averted by doing walk through yourself and contacting a plumber to fix the problem as soon as it arises.
3. During winter ensure that you drain your pipes of all water- When water freezes in pipes, they burst. To avoid this just drain off the water and turn off the valve.
4. Ensure that your plumbing is inspected regularly - This will be achieved by enlisting in a plumbing Chelsea MA service program.
5. Conserve your water- simple task like tightening your faucets and washing dishes in a closed sink will go a long way in lowering your water bill.


Last week, six men in Delhi died while cleaning a septic tank and a sewage pit in two separate incidents, bringing the issue of manual scavenging back into the national spotlight. A dehumanizing and exceedingly dangerous practice, manual scavenging—the removal of untreated human waste by predominantly Dalit workers—was banned in 1993. That law was amended in 2013 to include the cleaning of septic tanks and sewers. But five years later, both private companies and state and municipal authorities continue to flout the ban, depriving thousands of citizens of their right to live with dignity.
According to data collected by the National Commission for Safai Karamcharis, 123 people have died countrywide since 1 January 2017 while cleaning septic tanks and sewers. That's one death every five days. Given the lack of official data, even this is likely to be a gross underestimate. The Safai Karamchari Andolan (SKA), an NGO that works to rehabilitate manual scavengers, says they have documented 300 deaths due to this work in the same time period, and warns that the actual number is probably higher.


text:
package se.unlogic.standardutils.dao.querys;

import se.unlogic.standardutils.db.DBUtils;

import javax.sql.DataSource;
import java.sql.Connection;
import java.sql.ResultSet;
import java.sql.SQLException;
import java.util.Arrays;
import java.util.List;

public class UpdateQuery extends PreparedStatementQuery {

	private Integer affectedRows;

	public UpdateQuery(Connection connection, boolean closeConnectionOnExit, String query) throws SQLException {

		super(connection, closeConnectionOnExit, query);
	}

	public UpdateQuery(DataSource dataSource, boolean closeConnectionOnExit, String query) throws SQLException {

		super(dataSource, closeConnectionOnExit, query);
	}

	public void executeUpdate() throws SQLException {

		ResultSet rs = null;

		try {
			affectedRows = this.pstmt.executeUpdate();

		} catch (SQLException sqle) {
			throw sqle;
		} finally {
			DBUtils.closeResultSet(rs);
			DBUtils.closePreparedStatement(pstmt);

			if (this.closeConnectionOnExit) {
				DBUtils.closeConnection(connection);
			}

			this.closed = true;
		}
	}

	public void executeUpdate(List<GeneratedKeyCollector> keyCollectors) throws SQLException {

		ResultSet rs = null;

		try {
			affectedRows = this.pstmt.executeUpdate();

			if (affectedRows > 0) {
				rs = pstmt.getGeneratedKeys();

				if (rs.next()) {

					for (GeneratedKeyCollector keyCollector : keyCollectors) {

						keyCollector.collect(rs);
					}
				}
			}

		} catch (SQLException sqle) {
			throw sqle;
		} finally {
			DBUtils.closeResultSet(rs);
			DBUtils.closePreparedStatement(pstmt);

			if (this.closeConnectionOnExit) {
				DBUtils.closeConnection(connection);
			}

			this.closed = true;
		}
	}

	public void executeUpdate(GeneratedKeyCollector... keyCollectors) throws SQLException {

		executeUpdate(Arrays.asList(keyCollectors));
	}

	public Integer getAffectedRows() {

		return affectedRows;
	}
}


ArtStream welcomes adult and teen volunteers (ages 13+; rising 9th grade) for both one-time and ongoing opportunities in drama and social skills classes, Inclusive Theatre Companies, special events and at the ArtStream office. All volunteers must complete the volunteer application, meet the minimum age requirement, and be willing to donate at least 10 hours of service to ArtStream. Volunteers are eligible for SSL hours and will receive training. Please email volunteers@art-stream.org with any questions.
ArtStream will contact you to discuss your application.
Review orientation materials before attending your first assignment.
Please select your first choice. Volunteer options for this choice will automatically populate below.
No rehearsals on holidays. Some planned absences are acceptable.
In ArtStream's inclusive performing companies, actors with disabilities develop performances based on their own ideas. The final production is designed to showcase each actor's unique talents.
The inclusive performing companies are led by a professional team and are supported by volunteer mentors. Mentors perform onstage with the actors and support the actors both on and offstage.
The actors are always the focus onstage. Volunteer mentors are there to help them perform at their best!
I want to be an onstage volunteer mentor.
Assist with scene changes, costumes, or run a sound or light board during the two-week performance run of an ArtStream Inclusive Theatre original musical. The backstage volunteers report two the Stage Managers and have only limited contact with the actors.
Experience working backstage is a plus!
I want to volunteer backstage.
In ArtStream's Super Social Saturday workshops adults with intellectual and developmental disabilities (IDD) — including autism — have a dance party and practice social skills. We meet in Germantown, MD or in Oakton, VA.
Class volunteers at Super Social Saturday model good behavior and learn as peers to the participants. No experience necessary.
ArtStream classes are designed to teach communication, social and teamwork skills. Classes are taught by professional teaching artists.
I want to volunteer at Super Social Saturday!
In ArtStream's movement and music classes, adults with intellectual and developmental disabilities (IDD) — including autism — get in the groove! We meet in North Bethesda, MD in partnership with KEEN Greater DC.
Class volunteers model good behavior and learn as peers to the participants. No experience necessary.
I want to volunteer at Movement and Music.
In ArtStream's inclusive classes people with intellectual and developmental disabilities (IDD) — including autism — learn new acting skills like improv, dancing, and Shakespeare! ArtStream also offers social skills classes where participants practice skills like making a new friend, phone conversations, and hosting a party.
Class volunteers model good behavior and learn as peers to the participants. Volunteers don't have to be skilled actors or social butterflies. They just have to listen, learn, and do their best. No experience necessary.
Class dates, locations, and topics vary. Submit this form and an ArtStream representative will be in contact to find a class that works for you. You can also browse our calendar to find current classes. Keep in mind that some classes may not have room for more volunteers.
I want to volunteer in a weeknight ArtStream class.
I want to volunteer in a weekday ArtStream class.
ArtStream needs volunteers to sell tickets, merchandise, and concessions. We also need ushers to help our guests find their seats. All volunteer assignments are at the discretion of ArtStream. Volunteers report to the House Manager.
Volunteers will receive free admission to the performance that they work.
Volunteers must pre-arrange with ArtStream - we do not accept walk-in volunteers!
I want to volunteer to sell tickets and concessions.
ArtStream welcomes volunteers of all abilities on a need basis. We do not have weekly or monthly opportunities.
Volunteers will be supervised by a member of the office staff.
Please add me to the weekly ArtStream email list!
Please add me to the mailing list for class brochures and newsletters.
Good news: ArtStream volunteers are eligible for SSL hours! Please be sure to provide an email and phone number for your parent or guardian as an emergency contact.