mTOR Signaling in Growth, Metabolism, and Disease.

Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

Antiviral Actions of Interferons

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Central to innate immunity is the sensing of pathogen-associated molecular patterns by cytosolic and membrane-associated receptors. In particular, DNA is a potent activator of immune responses during infection or tissue damage, and evidence indicates that, in addition to the membrane-associated Toll-like receptor 9, an unidentified cytosolic DNA sensor(s) can activate type I interferon (IFN) and other immune responses. Here we report on a candidate DNA sensor, previously named DLM-1 (also called Z-DNA binding protein 1 (ZBP1)), for which biological function had remained unknown; we now propose the alternative name DAI (DNA-dependent activator of IFN-regulatory factors). The artificial expression of otherwise IFN-inducible DAI (DLM-1/ZBP1) in mouse fibroblasts selectively enhances the DNA-mediated induction of type I IFN and other genes involved in innate immunity. On the other hand, RNA interference of messenger RNA for DAI (DLM-1/ZBP1) in cells inhibits this gene induction programme upon stimulation by DNA from various sources. Moreover, DAI (DLM-1/ZBP1) binds to double-stranded DNA and, by doing so, enhances its association with the IRF3 transcription factor and the TBK1 serine/threonine kinase. These observations underscore an integral role of DAI (DLM-1/ZBP1) in the DNA-mediated activation of innate immune responses, and may offer new insight into the signalling mechanisms underlying DNA-associated antimicrobial immunity and autoimmune disorders.

DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response

The 1921 discovery of insulin was a Big Bang from which a vast and expanding universe of research into insulin action and resistance has issued. In the intervening century, some discoveries have ma...

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Mechanisms of Insulin Action and Insulin Resistance

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic α-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic α-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin Nup133, the sensor corresponds to an exposed loop of a β-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.

A general amphipathic |[alpha]|-helical motif for sensing membrane curvature

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

The editing enzyme double-stranded RNA adenosine deaminase includes a DNA binding domain, Zalpha, which is specific for left-handed Z-DNA. The 2.1 angstrom crystal structure of Zalpha complexed to DNA reveals that the substrate is in the left-handed Z conformation. The contacts between Zalpha and Z-DNA are made primarily with the "zigzag" sugar-phosphate backbone, which provides a basis for the specificity for the Z conformation. A single base contact is observed to guanine in the syn conformation, characteristic of Z-DNA. Intriguingly, the helix-turn-helix motif, frequently used to recognize B-DNA, is used by Zalpha to contact Z-DNA.

http://science.sciencemag.org/content/sci/284/5421/1841.full.pdf

Crystal Structure of the Zα Domain of the Human Editing Enzyme ADAR1 Bound to Left-Handed Z-DNA

Eukaryotic cells coordinate growth with the availability of nutrients through the mechanistic target of rapamycin complex 1 (mTORC1), a master growth regulator. Leucine is of particular importance and activates mTORC1 via the Rag guanosine triphosphatases and their regulators GATOR1 and GATOR2. Sestrin2 interacts with GATOR2 and is a leucine sensor. Here we present the 2.7 angstrom crystal structure of Sestrin2 in complex with leucine. Leucine binds through a single pocket that coordinates its charged functional groups and confers specificity for the hydrophobic side chain. A loop encloses leucine and forms a lid-latch mechanism required for binding. A structure-guided mutation in Sestrin2 that decreases its affinity for leucine leads to a concomitant increase in the leucine concentration required for mTORC1 activation in cells. These results provide a structural mechanism of amino acid sensing by the mTORC1 pathway.

https://science.sciencemag.org/content/sci/351/6268/53.full.pdf

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the nuclear envelope and are composed of KASH and SUN proteins residing in the outer and inner nuclear membrane, respectively. LINC formation relies on direct binding of KASH and SUN in the perinuclear space. Thereby, molecular tethers are formed that can transmit forces for chromosome movements, nuclear migration, and anchorage. We present crystal structures of the human SUN2-KASH1/2 complex, the core of the LINC complex. The SUN2 domain is rigidly attached to a trimeric coiled coil that prepositions it to bind three KASH peptides. The peptides bind in three deep and expansive grooves formed between adjacent SUN domains, effectively acting as molecular glue. In addition, a disulfide between conserved cysteines on SUN and KASH covalently links both proteins. The structure provides the basis of LINC complex formation and suggests a model for how LINC complexes might arrange into higher-order clusters to enhance force-coupling.

Thomas U. Schwartz

Papers

A general amphipathic |[alpha]|-helical motif for sensing membrane curvature

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins

Crystal Structure of the Zα Domain of the Human Editing Enzyme ADAR1 Bound to Left-Handed Z-DNA

Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway

LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins