Hexokinase-II (HK-II) catalyzes the first step of glycolysis and also functions as a protective molecule, however, its role in protective autophagy has not been determined. Results showed that inhibition of HK-II diminished, while overexpression of HK-II potentiated, autophagy induced by glucose deprivation in cardiomyocyte and non-cardiomyocyte cells. Immunoprecipitation studies revealed that HK-II binds to and inhibits the autophagy suppressor, mTOR complex 1 (TORC1), and this binding was increased by glucose deprivation. The TOS motif, a scaffold sequence responsible for binding TORC1 substrates, is present in HK-II and mutating it blocked its ability to bind to TORC1 and regulate protective autophagy. The transition from glycolysis to autophagy appears to be regulated by a decrease in glucose-6 phosphate. We suggest that HK-II binds TORC1 as a decoy substrate and provides a previously unrecognized mechanism for switching cells from a metabolic economy based on plentiful energy, to one of conservation, under starvation.
Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homoeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here we report that lysine acetylation of PDHA1 and PDP1 is common in EGF-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1 and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important to promote glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct post-translational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.
The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially-regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. 21/24 non-small-cell-lung carcinomas we analyzed, express IWS1. More important, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern, and with Akt phosphorylation and Akt3 expression. These data identify a novel Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer.
Akt isoforms; PKB; IWS1; SETD2; MRG15; PTB; FGFR2; phosphoproteomics; RNA metabolism; alternative splicing; histone H3 K36 trimethylation; transcriptional elongation
The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here we demonstrate a novel mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine148 in the luminal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyper-respond to ER stress with sustained auto-phosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated up-regulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.
In this issue, Roberts et al. (2014) describe how hexokinase and mTORC1 cooperate to sense disequilibrium between glucose uptake and utilization and direct the balance of anabolism and catabolism to ensure the appropriate use of cellular resources.
Genomic analysis has revealed the existence of a large number of long non-coding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Cells display dramatic changes of gene expression upon environmental changes. Upon osmostress, hundreds of stress-responsive genes are induced by the stress-activated protein kinase (SAPK) p38/Hog1. Using whole-genome tiling arrays, we found that Hog1 induces a set of lncRNAs upon stress. One of the genes expressing a Hog1-dependent lncRNAs in antisense orientation is CDC28, the CDK1 kinase that controls the cell cycle in yeast. Cdc28 lncRNA mediates the establishment of gene looping and the relocalization of Hog1 and RSC from the 3′UTR to the +1 nucleosome to induce CDC28 expression. The increase in the levels of Cdc28 results in cells able to re-enter the cell cycle more efficiently after stress. This may represent a general mechanism to prime expression of genes needed after stresses are alleviated.
Eukaryotic chromosomes are partitioned into topologically associating domains (TADs) that are demarcated by distinct insulator-binding proteins (IBPs) in Drosophila. Whether IBPs regulate specific long-range contacts and how this may impact gene expression remains unclear. Here we identify ‘indirect peaks’ of multiple IBPs, that represent their distant sites of interactions through long-range contacts. Indirect peaks depend on protein-protein interactions among multiple IBPs and their common co-factors, including CP190, as confirmed by high-resolution analyses of long-range contacts. Mutant IBPs unable to interact with CP190 impair long-range contacts as well as the expression of hundreds of distant genes that are specifically flanked by indirect peaks. Regulation of distant genes strongly correlates with RNAPII pausing, highlighting how this key transcriptional stage may trap insulator-based long-range interactions. Our data illustrate how indirect peaks may decipher gene regulatory networks through specific long-range interactions.
Insulators; Long-range interactions; RNA Polymerase II pausing; Higher-order Chromatin organization; Chromosome domains; Physical borders; Transcription Factories; Topological Domains
We have solved two families of crystal structures of human Dicer ‘platform-PAZ-connector helix’ cassette in complex with siRNAs. The structures possess two adjacently positioned pockets: a 2-nucleotide 3′-overhang-binding pocket within the PAZ domain (3′-pocket) and a newly identified phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contain a knob-like α-helical protrusion, designated ‘hDicer-specific helix’, that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned towards the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5′-phosphate from the phosphate pocket, while retaining the 3′-overhang in the 3′-pocket.
RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.
Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multi-subunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here we determined the crystal structure of the N-terminal region (599–627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3–H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.
The intrinsically disordered yeast protein Sem1 (DSS1 in mammals) participates in multiple protein complexes, including the proteasome, but its role(s) within these complexes is uncertain. We report that Sem1 enforces the ordered incorporation of subunits Rpn3 and Rpn7 into the assembling proteasome lid. Sem1 uses conserved acidic segments separated by a flexible linker to grasp Rpn3 and Rpn7. The same segments are used for protein binding in other complexes, but in the proteasome lid they are uniquely deployed for recognizing separate polypeptides. We engineered TEV protease-cleavage sites into Sem1 to show that the tethering function of Sem1 is important for the biogenesis and integrity of the Rpn3-Sem1-Rpn7 ternary complex but becomes dispensable once the ternary complex incorporates into larger lid precursors. Thus, although Sem1 is a stoichiometric component of the mature proteasome, it has a distinct, chaperone-like function specific to early stages of proteasome assembly.
Tudor domain containing protein 3 (TDRD3) is a major methyl-arginine effector molecule that “reads” methyl-histone marks and facilitates gene transcription. However, the underlying mechanism by which TDRD3 functions as a transcriptional coactivator is unknown. We identified topoisomerase IIIB (TOP3B) as a component of the TDRD3 complex. TDRD3 serves as a molecular bridge between TOP3B and arginine-methylated histones. The TDRD3-TOP3B complex is recruited to the c-MYC gene promoter primarily by the H4R3me2a mark, and the complex promotes c-MYC gene expression. TOP3B relaxes negative supercoiled DNA and reduces transcription-generated R-loops in vitro. TDRD3 knockdown in cells increases R-loop formation at the c-MYC locus, and Tdrd3-null mice exhibit elevated R-loop formation at this locus in B cells. Tdrd3-null mice show significantly increased c-Myc/Igh translocation, a process driven by R-loop structures. By reducing negative supercoiling and resolving R-loop, TOP3B promotes transcription, protects against DNA damage and reduces the frequency of chromosomal translocations.
In this issue, Reddy et al (2014) reveal a new twist in the molecular mechanism leading to p53 activation upon cellular stress, illuminating an unexpected nuclear role for a nucleotide biosynthetic enzyme in regulation of a potent tumor suppressor.
The displacement loop (D-loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, Rad51 recombinase is assisted in D-loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D-loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover novel features of D-loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a newly discovered single-stranded DNA binding domain. We present a unified model where Rad54 acts as an hDNA pump, which drives D-loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.
During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin’s N-terminal domain, while Listerin’s C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control.
•Targeting of Listerin E3 ligase to translationally stalled proteins requires NEMF•NEMF binds to the 60S subunit, prevents 40S rejoining, and stabilizes Listerin•Cryo-EM structure of the 60S-NEMF-Listerin complex reveals a network of interactions•NEMF discriminates 60S-nascent chains from empty 60S via exposed peptidyl tRNA
Ribosomes that stall during translation are dissociated, and the aberrant polypeptide within the 60S ribosomal subunit is poly-ubiquitinated by Listerin. Shao et al. identify NEMF as a factor that facilitates Listerin recruitment to 60S-nascent chains and provide structural insights into how targets are selected during this ribosome-associated quality control pathway.
DNA methylation patterns are dynamically controlled by DNA methylation and active DNA demethylation, but the mechanisms of regulation of active DNA demethylation are not well understood. Through forward genetic screens for Arabidopsis mutants showing DNA hypermethylation at specific loci and increased silencing of reporter genes, we identified IDM2 (increased DNA methylation 2) as a regulator of DNA demethylation and gene silencing. IDM2 dysfunction causes DNA hypermethylation and silencing of reporter genes and some endogenous genes. These effects of idm2 mutations are similar to those of mutations in IDM1, a regulator of active DNA demethylation. IDM2 encodes an α-crystallin domain protein in the nucleus. IDM2 and IDM1 interact physically and partially colocalize at discrete subnuclear foci. IDM2 is required for the full activity of H3K18 acetylation but not H3K23 acetylation of IDM1 in planta. Our results suggest that IDM2 functions in active DNA demethylation and in antisilencing by regulating IDM1.
SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.
•Prokaryotic Smc-ScpAB complexes form rod-like structures•Binding of ATP and DNA induces a rod-to-ring transition in prokaryotic condensin•The condensin hinge is rigidly anchored to its coiled coil•The rod-like conformation is a conserved feature of SMC protein dimers
Soh et al. show that the rod-like conformation is a conserved architectural scheme of SMC complexes. Upon ATP-induced binding to DNA, the juxtaposed coiled coils of prokaryotic Smc-ScpAB adopt an open conformation to expose a DNA binding site at the inner surface of the hinge domain.
Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles.
•PI(5)P positively regulates autophagy•PI(5)P is associated with autophagy effectors that bind PI(3)P•PI(5)P sustains noncanonical autophagy in PI(3)P-depleted cells•PI(5)P is essential for VPS34-independent, glucose-starvation-induced autophagy
PI(3)P, the product of VPS34, regulates canonical autophagy; however, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Vicinanza et al. show that PI(5)P can regulate autophagy, even in cells where VPS34 is compromised and acts via PI(3)P effectors. This provides a mechanistic explanation for forms of noncanonical autophagy.