SUMMARY Metformin has utility in cancer prevention and treatment, though the mechanisms for these effects remain elusive. Through genetic screening in C. elegans, we uncover two metformin response elements: the nuclear pore complex (NPC) and acyl-CoA dehydrogenase family member-10 (ACAD10). We demonstrate that biguanides inhibit growth by inhibiting mitochondrial respiratory capacity, which restrains transit of the RagA-RagC GTPase heterodimer through the NPC. Nuclear exclusion renders RagC incapable of gaining the GDP-bound state necessary to stimulate mTORC1. Biguanide-induced inactivation of mTORC1 subsequently inhibits growth through transcriptional induction of ACAD10. This ancient metformin response pathway is conserved from worms to humans. Both restricted nuclear pore transit and upregulation of ACAD10 are required for biguanides to reduce viability in melanoma and pancreatic cancer cells, and to extend C. elegans lifespan. This pathway provides a unified mechanism by which metformin kills cancer cells and extends lifespan, and illuminates potential cancer targets.
Like orthologues in other members of the subfamily Alphaherpesvirinae, the U S 3 gene of herpes simplex virus type 1 (HSV-1) encodes a serine/threonine kinase (13, 37). These proteins have been implicated in nuclear egress, prevention of apoptosis, and modulation of the actin cytoskeleton to promote the cell-to-cell spread of virions (11,14,19,22,25,42,44,46,52). The current study focuses on the role of the U S 3 gene-encoded kinase activity in nuclear egress of nucleocapsids and virions.Although models of HSV virion egress differ as to the extent of its contribution, all models propose that at some point during the course of infection with wild-type herpesviruses, nucleocapsids assemble in the nucleoplasm and bud through the inner nuclear membrane (INM) and into the perinuclear space (20,51,53). This compartment is delimited by the INM and outer nuclear membrane (ONM) and is continuous with the lumen of the endoplasmic reticulum. To become enveloped, capsids must bypass the nuclear lamina, a fibrous meshwork lining the nucleoplasmic face of the INM. The nuclear lamina provides structural rigidity to the nucleus and is essential for transcription and DNA replication (15,48). The lamina contains a series of type 5 intermediate filaments composed of lamin types A, B1, B2, and C; types A and C are products of RNA splice variants from the LmnA transcript, whereas types B1 and B2 are derived from other genes (12,16,17). Like all intermediate filaments, lamins comprise globular head and tail domains that flank a rod domain (12). The rod domains of two lamins intertwine to form protomers, whereas regions bordering the rod/head and rod/tail domains likely interact with other lamin protomers to form longer filaments (40). The globular domains interact with a variety of proteins in the lamina and INM.One remarkable feature of the lamina is its dynamic nature. The lamina expands by the addition of protomers during interphase, is completely disassembled prior to mitosis, and is partially disrupted during apoptosis. Phosphorylation likely plays a role in lamin dynamics in all phases of the cell cycle. The disassembly of the lamina during mitosis is associated with phosphorylation of lamin A/C by cdc2 kinase at Ser 390 and Ser 392 and during apoptosis by protein kinase C (PKC) delta (5,7,8,32,33). Protein kinase C can phosphorylate lamin A/C at Ser 572 in vitro (8).The architecture of the nuclear lamina is altered from its normal state during HSV-1 infection (3,31,40,47,49). Depending on the cell line and time after infection, these changes include (i) limited displacement and conformational changes of lamin A/C (3, 40, 49), (ii) redistribution of lamin B to a perinuclear region (31,47), and (iii) increased mobility and mislocalization of lamin B receptor, one of several integral membrane proteins that anchor the nuclear lamina to the INM (47, 48). In attempts to understand the mechanism(s) by which * Corresponding author. Mailing address:
The U L 31 and U L 34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26,27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26,28). pU L 34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU L 31 and holds the latter in close approximation to the INM (16,19,26,31,36,37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU L 31 and pU L 34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U S 3 protein is a promiscuous kinase that phosphorylates pU L 31, pU L 34, and several other viral and cellular components (1,2,5,11,15,(21)(22)(23)25). In the absence of pU S 3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU L 31/pU L 34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12,14,27,29), and (iii) the onset of infectious virus production is delayed (21,29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U S 3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3,4,9,10,18,30,33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU S 3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU S 3 kinase responsible for the altered localization of the pU L 31/pU L 34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU L 34 was not responsible for the nuclear egress defects induced by the absence of pU S 3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU S 3-mediated phosphorylation of pU L 31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U S 3 kinase-dead phenotype, including the ...
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