Glycogen is the primary source of storage carbohydrate in mammals. Glycogen synthesis and degradation either consumes or produces glucose‐6‐phosphate (G6P), a key metabolite essential for central carbon metabolism. Several studies have reported glycogen accumulation in specific sub‐cellular organelles, suggesting that glycogen localization is not random. Nuclear glycogen was first reported in the 1890s in hepatocytes, and subsequent reports identified glycogen accumulation near the ER, and mitochondria. Cumulatively, these data suggest compartment‐specific roles for glycogen that have yet to be fully elucidated. The E3 ubiquitin ligase malin is a modulator of glycogen architecture through ubiquitination of multiple glycogen metabolic enzymes. Our previous work and that of others demonstrated that malin activity is required for normal glycogen architecture as somatic loss‐of‐function mutations in malin results in aberrant glycogen. We report that decreased levels of malin in human NSCLC samples results in aberrant accumulation of nuclear and cytoplasmic glycogen followed by glucose metabolism. We employed ultra‐pure nuclear preparations coupled with stable isotope technology to define the origin and biological destiny of nuclear and cytoplasmic glycogen. We show that malin is required to maintain compartmentalized glycogen metabolism, and loss of malin in NSCLC results in 1) the accumulation of nuclear glycogen and reduced histone acetylation in the nucleus, and 2) aberrant accumulation of cytoplasmic glycogen accompanied by increased glucose central carbon metabolism. Re‐expression of malin in model NSCLC cell lines, normalized both nuclear and cytoplasmic glycogen, and decreased glucose metabolism both in vitro and in vivo. Finally, malin expression blunted in vivo xenograft growth of model NSCLC cell lines. These data provide new insights for an alternative signaling pathway that regulates metabolism in NSCLC to drive tumor progression.

The role of cellular metabolites in the direct control of signaling is an emerging and rapidly evolving field. Herein, we identify a key role for nuclear glycogen in epigenetic regulation through compartmentalized acetyl CoA production and histone acetylation. Nuclear glycogenolysis is dependent on ubiquitination and translocation of glycogen phosphorylase (GP) into the nucleus by malin, an E3 ubiquitin ligase. We developed an innovative in organello stable isotope tracer method coupled to mass spectrometry analysis to define the metabolic fate of nuclear glycogen. This work revealed that GP is required for nuclear glycogen degradation and subsequent glycolysis to generate substrates for histone acetylation. Inhibition of nuclear glycogenolysis is found to be particularly important in non-small cell lung cancer (NSCLC), as evident by increased nuclear glycogen accumulation and malin suppression in NSCLC. Re-introduction of malin in model NSCLC cell lines restores nuclear glycogenolysis, resulting in increased histone acetylation and transcriptional changes that delay cancer cell growth in vivo . This study uncovers a previously unknown role for glycogen metabolism in the nucleus and elucidates another way by which cellular metabolites control epigenetic regulation.

Nuclear glycogen was first documented in the early 1940s, but its role in cellular physiology remained elusive. In this study, we utilized pure nuclei preparations and stable isotope tracers to define the origin and metabolic fate of nuclear glycogen. Herein, we describe a key function for nuclear glycogen in epigenetic regulation through compartmentalized pyruvate production and histone acetylation. This pathway is altered in human non-small cell lung cancers, as surgical specimens accumulate glycogen in the nucleus. We demonstrate that the decreased abundance of malin, an E3 ubiquitin ligase, impaired nuclear glycogenolysis by preventing the nuclear translocation of glycogen phosphorylase and causing nuclear glycogen accumulation. Re-introduction of malin in lung cancer cells restored nuclear glycogenolysis, increased histone acetylation, and decreased growth of cancer cells transplanted into mice. This study uncovers a previously unknown role for glycogen metabolism in the nucleus and elucidates another mechanism by which cellular metabolites control epigenetic regulation.

Sucrose non-fermenting 1-related protein kinase 1 (SnRK1) is a central metabolic regulator and the plant orthologue of the mammalian AMP-activated protein kinase (AMPK); both are energy-sensing heterotrimeric enzymes comprising a catalytic α- and regulatory β- and γ-subunits. α-Subunits contain a serine/threonine kinase domain (KD) at their N-terminus that is immediately followed by a small regulatory domain termed the auto-inhibitory domain (AID) in AMPK and the ubiquitin-associated domain (UBA) in SnRK1. Association of the AID with the AMPK KD inhibits activating phosphorylation of the KD by upstream kinases and promotes dephosphorylation, as well as inhibiting AMPK catalytic activity. Despite these mechanistic insights regarding the AMPK AID, the SnRK1 UBA regulatory implications have not been investigated. Using recombinant protein comprising either the KD-only or KD-AID/KD-UBA, we found that the UBA of SnRK1 acts in a distinct regulatory manner to its orthologous AID of AMPK. Firstly, the plant upstream kinase GRIK2 preferentially phosphorylates the SnRK1 KD-UBA. Secondly, the SnRK1 KD in the absence of the UBA shows near identical initial catalytic activity to the KD-UBA, but in comparison a rapid loss of catalytic activity is observed. Our findings indicate that the role of the UBA in SnRK1 regulation may be more akin to that of the UBA in the mammalian AMPK-related kinases rather than its immediate functional orthologue, AMPK. This study adds to a growing body of work demonstrating the divergent regulatory mechanisms of the orthologous plant SnRK1 and mammalian AMPK.

The growth of the bioethanol industry over the last decade has been a result of the growing demand for gasoline fuels, which in the United States constitutes at least 10% by volume, and the implementation of higher Renewable Fuel Standards (RFS). Bioethanol is produced by the fermentation of simple sugars by yeasts and as a result starch feedstocks must undergo pretreatment steps. The various mechanical, thermal and chemical treatments used to degrade starch are both expensive and energy intensive. Alternatively, plants use a reversible phosphorylation mechanism to solubilize starch and access stored glucose for nocturnal metabolism. Starch is formed by linear α‐1,4 linked glucose units and branching glucose via α‐1,6 bonds which form insoluble helical structures. Starch kinases phosphorylate the crystalline layers, disrupting the helical structures, and exposing the polymer to hydrolysis by amylases. This mechanism could be adopted during biofuels production to decrease the need for solubilizing treatments. Starch kinases are characterized by two distinct domains: carbohydrate binding modules (CBMs) and a dikinase domain connected via a peptide linker. The CBM functions to locate the kinase on the starch substrate, which is subsequently phosphorylated by the kinase domain. In order to expand the substrate affinity of a kinase, we fused the CBM from a Neurospora crassa starch‐active polysaccharide monooxygenase to multiple dikinase domains. We defined the melting temperature of the chimeric proteins using differential scanning fluorimetry and found that the chimeric proteins had increased stability. Using [33P‐β‐]ATP, we radiolabeled starch with the chimeric proteins and found that they exhibited at least 7‐fold greater activity than wild type dikinase using maize, barley, and potato starch as the substrate.Support or Funding InformationNSF IIA‐1355438 (R. Andrews), NSF MCB‐1252345 (M. Gentry)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase (AMPK) in mammals, SnRK1 acts protectively to maintain homeostasis in the face of fluctuations in energy status. Despite a conserved function, the structure and regulation of the plant kinase differ considerably from its relatively well-understood opisthokont orthologues. In this review, we highlight the known plant-specific modes of regulation involving SnRK1 together with new insights based on a 3D molecular model of the kinase. We also summarise how these differences from other orthologues may be specific adaptations to plant metabolism, and offer insights into possible avenues of future inquiry into this enigmatic enzyme.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic β and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, β(3) and βγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique βγ subunit and one of three non-catalytic β subunits: β(1), β(2) or the plant-specific β(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical βγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the β(1), β(2) and βγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

AMP‐activated protein kinase (AMPK) is a heterotrimer of catalytic (α) and regulatory (β and γ) subunits with at least two isoforms for each subunit. AMPK β1 is widely expressed whilst AMPK β2 is highly expressed in muscle and both β isoforms contain a mid‐molecule carbohydrate‐binding module (β‐CBM). Here we show that β2‐CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single α‐1,6 branched residue. Deletion of Thr‐101 reduces affinity for single α‐1,6 branched oligosaccharides by 3‐fold, while insertion of this residue into the equivalent position in the β1‐CBM sequence increases affinity by 3‐fold, confirming the functional importance of this residue.