Cancer cells need to meet the metabolic demands of rapid cell growth within a continually changing microenvironment. Genetic mechanisms for reprogramming cellular metabolism toward proliferative, pro-survival pathways are well-reported. However, post-translational mechanisms, which would enable more rapid, reversible adaptations of cellular metabolism in response to protein signaling or environmental sensing systems, are less well understood. Here we demonstrate that the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a key metabolic regulator of glucose metabolism. O-GlcNAc is dynamically induced at Ser529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibits PFK1 activity and redirects the flux of glucose from glycolysis through the pentose phosphate pathway (PPP), thereby conferring a selective growth advantage to cancer cells. Blocking glycosylation of PFK1 at Ser529 reduced cancer cell proliferation in vitro and impaired tumor formation in vivo. These studies reveal an unexpected mechanism for the regulation of metabolic enzymes and pathways, and pinpoint a new therapeutic approach for combating cancer.

Summary The volume-regulated anion channel (VRAC) is activated when a cell swells, and plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC, and how the channel is gated by cell swelling are unknown. Here we show that SWELL1 with up to four other LRRC8 subunits assemble into heterogeneous complexes of ~800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ), in the absence of an osmotic gradient, activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore, and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ.

Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3+2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct ingel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of OGlcNAc.Understanding posttranslational modifications to proteins is critical for elucidating the functional roles of proteins within the dynamic environment of cells. O-Linked β-Nacetylglucosamine (O-GlcNAc) glycosylation has emerged as important for the regulation of diverse cellular processes, including transcription, cell division, and glucose homeostasis. 1 While new chemical tools have provided rapid, sensitive methods for detecting the modification and enabled better control over the activity of O-GlcNAc enzymes, 1a,2 significant challenges remain with regard to elucidating the functions of O-GlcNAc in cells. For instance, a robust method for the direct fluorescence detection of O-GlcNAc proteins in gels would permit monitoring of changes in glycosylation levels in response to cellular stimuli and greatly extend the reach of existing technologies. Furthermore, new tools for imaging OGlcNAc-glycosylated proteins would enable the expression and dynamics of the modification to be monitored in cells and tissues. Here, we report an advanced chemoenzymatic labeling strategy that addresses these important needs. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptPrevious studies have shown that an engineered β-1,4-galactosyltransferase enzyme (Y289L GalT) efficiently transfers a ketogalactose moiety from an unnatural UDP substrate selectively onto O-GlcNAc-modified proteins. 2a However, treatment of cell lysates with an aminooxy fluorescein derivative resulted in some nonspecific labeling of proteins. We therefore investigated whether Y289L GalT would accept the UDP-azidogalactose substrate 1 (UDPGalNAz), which would allow for labeling of O-GlcNAc proteins using [3+2] azide-alkyne cycloaddition chemistry ( Figure 1A). 3 In addition to providing alternative dyes to potentially reduce nonspecific interactions, this Cu(I)-catalyzed cycloaddition reaction would have the advantage of being performed more rapidly and at physiological pH.We tested the approach using α-crystallin, a known O-GlcNAc-modified protein with a low extent (~10%) of glycosylation. α-Crystallin was treated with 1 and Y289L GalT, followed by reaction with CuSO 4 , sodium ascor...