Search

Skeletal muscle atrophy is caused by various conditions, including aging, disuse related to a sedentary lifestyle and lack of physical activity, and cachexia. Our insufficient understanding of the molecular mechanism underlying muscle atrophy limits the targets for the development of effective pharmacologic treatments and preventions. Here, we identified Krüppel-like factor 5 (KLF5), a zinc-finger transcription factor, as a key mediator of the early muscle atrophy program. KLF5 was up-regulated in atrophying myotubes as an early response to dexamethasone or simulated microgravity in vitro. Skeletal muscle-selective deletion of Klf5 significantly attenuated muscle atrophy induced by mechanical unloading in mice. Transcriptomeand genome-wide chromatin accessibility analyses revealed that KLF5 regulates atrophy-related programs, including metabolic changes and E3-ubiquitin ligase-mediated proteolysis, in coordination with Foxo1. The synthetic retinoic acid receptor agonist Am80, a KLF5 inhibitor, suppressed both dexamethasone- and microgravityinduced muscle atrophy in vitro and oral Am80 ameliorated disuse- and dexamethasone-induced atrophy in mice. Moreover, in three independent sets of transcriptomic data from human skeletal muscle, KLF5 expression significantly increased with age and the presence of sarcopenia and correlated positively with the expression of the atrophy-related ubiquitin ligase genes FBXO32 and TRIM63. These findings demonstrate that KLF5 is a key transcriptional regulator mediating muscle atrophy and that pharmacological intervention with Am80 is a potentially preventive treatment. [ABSTRACT FROM AUTHOR]

The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)-XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking. [ABSTRACT FROM AUTHOR]

RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA. [ABSTRACT FROM AUTHOR]

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi. [ABSTRACT FROM AUTHOR]

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. Lossof- function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1. Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly. [ABSTRACT FROM AUTHOR]

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs and thus, the reported synthetic principles will find broad applicability. [ABSTRACT FROM AUTHOR]

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization. [ABSTRACT FROM AUTHOR]

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation-libration-screw (TLS), liquid- like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering. [ABSTRACT FROM AUTHOR]

Proteins achieve their complex functions, such as molecular recognition with high affinity and specificity, through intricate threedimensional geometries in functional sites. To engineer new protein functions, accurate positioning of amino acid functional groups is therefore critical but has remained difficult to achieve by computational methods because of current limitations in the design of new conformations with arbitrary user-defined geometries. Here, we introduce two computational methods capable of generating and predicting new local protein geometries: fragment kinematic closure (FKIC) and loophash kinematic closure (LHKIC). FKIC and LHKIC integrate two approaches: robotics-inspired kinematics of protein conformations and insertion of peptide fragments. We show that FKIC and LHKIC predict native-like conformations at atomic accuracy and with up to 140-fold improvements in sampling efficiency over previous approaches. We then use these methods to create a design protocol, pull into place (PIP), to position functionally important side chains via design of backbone conformations. We validate PIP by remodeling a sizeable active site region in an enzyme and confirming the engineered new conformations of two designs with crystal structures. The described methods can be applied broadly to the design of userdefined geometries for new protein functions. [ABSTRACT FROM AUTHOR]

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis. [ABSTRACT FROM AUTHOR]

Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as "cross-correction." Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease. [ABSTRACT FROM AUTHOR]

The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is the penultimate step of chlorophyll biosynthesis. In oxygenic photosynthetic bacteria, algae, and plants, this reaction can be catalyzed by the light-dependent Pchlide oxidoreductase (LPOR), a member of the short-chain dehydrogenase superfamily sharing a conserved Rossmann fold for NAD(P)H binding and the catalytic activity. Whereas modeling and simulation approaches have been used to study the catalytic mechanism of this light-driven reaction, key details of the LPOR structure remain unclear. We determined the crystal structures of LPOR from two cyanobacteria, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus. Structural analysis defines the LPOR core fold, outlines the LPOR-NADPH interaction network, identifies the residues forming the substrate cavity and the proton-relay path, and reveals the role of the LPOR-specific loop. These findings provide a basis for understanding the structurefunction relationships of the light-driven Pchlide reduction. [ABSTRACT FROM AUTHOR]

High-income countries often outsource material demands to poorer countries along with the associated environmental damage. This phenomenon can also occur within (large) countries, such as China, which was responsible for 24 to 30% of the global material footprint (MF) between 2007 and 2010. Understanding the distribution and development of China's MF is hence critical for resource efficiency and circular economy ambitions globally. Here we present a comprehensive analysis of China's MF at the provincial and sectoral levels. We combine provincial-level input-output data with sector- and province-specific trade data, detailed material extraction data, and the global input-output database EXIOBASE. We find that some provinces haveMFs equivalent to medium-sized, highincome countries and limited evidence of material decoupling. Lower-income regions with high levels of material extraction can have an MF per capita as large as developed provinces due to much higher material intensities. The higher-income south-coastal provinces have lower MF per capita than equally developed provinces. This finding relates partly to differences in economic structure but indicates the potential for improvement across provinces. Investment via capital formation is up to 4 times more resource-intensive than consumption and drives 49 to 86% of provincial-level MFs (the Organisation for Economic Co-operation and Development average is 37%). Resource-efficient production, efficient use of capital goods/infrastructure, and circular design are essential for reductions in China's MF. Policy efforts to shift to a high-quality development model may reduce material intensities, preferably while avoiding the further outsourcing of high-intensity activities to other provinces or lower-income countries. [ABSTRACT FROM AUTHOR]

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDPGlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAc- Man3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GTA Rossmann-like fold that employs conserved divalent cationdependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate "recognition arm." Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells. [ABSTRACT FROM AUTHOR]

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC self-renewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs. [ABSTRACT FROM AUTHOR]